Variant alpha-amylases from Bacillus subtilis and methods of uses, thereof

ABSTRACT

Alpha-amylases from  Bacillus subtilis  (AmyE), variants thereof, nucleic acids encoding the same, and host cells comprising the nucleic acids are provided. Methods of using AmyE or variants thereof are disclosed, including liquefaction and/or saccharification of starch. Such methods may yield sugars useful for ethanol production or high fructose corn syrup production. In some cases, the amylases can be used at low pH, in the absence of calcium, and/or in the absence of a glucoamylase.

PRIORITY

The present application is a divisional application of U.S. patentapplication Ser. No. 12/479,427, filed Jun. 5, 2009, now U.S. Pat. No.8,323,945 which claims priority to U.S. Provisional Patent ApplicationNos. 61/059,513, filed Jun. 6, 2008, and 61/059,618, filed on Jun. 6,2008, which are incorporated by reference in their entireties.

FIELD OF THE INVENTION

Alpha-amylases from Bacillus subtilis (AmyE), variants thereof, nucleicacids encoding the same, and host cells comprising the nucleic acids areprovided. Methods of using AmyE or variants thereof are disclosed,including liquefaction and/or saccharification of starch. Such methodsmay yield sugars useful for ethanol production or high fructose cornsyrup production. In some cases, the amylases can be used at low pH, inthe absence of calcium, and/or in the absence of a glucoamylase.

BACKGROUND

Grain, cereal, and vegetable starches, e.g., cornstarch, are widely usedin the industrial manufacture of products such as syrups and biofuels.For example, high fructose corn syrup (HFCS) is a processed form of cornsyrup having high fructose content and a sweetness comparable to sugar,making HFCS useful as a sugar substitute in soft drinks and otherprocessed foods. HFCS production currently represents a billion dollarindustry. The production of ethanol as a biofuel is also a growingindustry.

Syrups and biofuels can be produced from starch by an enzymatic processthat catalyzes the breakdown of starch into glucose. This enzymaticprocess typically involves a sequence of enzyme-catalyzed reactions:

(1) Liquefaction: Alpha (α)-amylases (EC 3.2.1.1) first catalyze thedegradation of a starch suspension, which may contain 30-40% w/w drysolids (ds), to maltodextrans. α-amylases are endohydrolases thatcatalyze the random cleavage of internal α-1,4-D-glucosidic bonds.Because liquefaction typically is conducted at high temperatures, e.g.,90-100° C., thermostable α-amylases, such as an α-amylase from Bacillussp., are preferred for this step. α-Amylases currently used for thisstep, e.g., α-amylases from B. licheniformis, B. amyloliquefaciens, andB. stearothermophilus (AmyS), do not produce significant amounts ofglucose. Instead, the resulting liquefact has a low dextrose equivalent(DE) and contains maltose and sugars with high degrees of polymerization(DPn).

(2) Saccharification: Glucoamylases and/or maltogenic α-amylasescatalyze the hydrolysis of non-reducing ends of the maltodextrans formedafter liquefaction, releasing D-glucose, maltose and isomaltose.Saccharification produces either glucose-rich or high-maltose syrups. Inthe former case, glucoamylases typically catalyze saccharification underacidic conditions at elevated temperatures, e.g., 60° C., pH 4.3.Glucoamylases used in this process typically are obtained from fungi,e.g., Aspergillus niger glucoamylase used in OPTIDEX® L400 or Humiculagrisea glucoamylase. De-branching enzymes, such as pullulanases, can aidsaccharification.

Maltogenic α-amylases alternatively may catalyze saccharification toform high-maltose syrups. Maltogenic α-amylases typically have a higheroptimal pH and a lower optimal temperature than glucoamylase, andmaltogenic amylases typically require Ca²⁺. Maltogenic α-amylasescurrently used for this application include B. subtilis α-amylases,plant amylases, and the α-amylase from Aspergillus oryzae, the activeingredient of CLARASE® L. Exemplary saccharification reactions used toproduce various products are depicted below:

(3) Further processing: A branch point in the process occurs after theproduction of a glucose-rich syrup, shown on the left side of thereaction pathways above. If the final desired product is a biofuel,yeast can ferment the glucose-rich syrup to ethanol. On the other hand,if the final desired product is a fructose-rich syrup, glucose isomerasecan catalyze the conversion of the glucose-rich syrup to fructose.

Saccharification is the rate-limiting step in the production of aglucose-rich syrup. Saccharification typically occurs over 48-72 hours,by which time many fungal glucoamylases lose significant activity.Further, although maltogenic α-amylases and glucoamylases both cancatalyze saccharification, the enzymes typically operate at differentoptimal pH and temperatures, as shown above. If both enzymes are usedsequentially, the difference in reaction conditions between the twoenzymes necessitates adjusting the pH and temperature, which slows downthe overall the process and may give rise to the formation of insolubleamylose aggregates.

Accordingly, there is a need in the art for an improved process ofmaking industrial products from starch. In particular, there is a needfor improved efficiencies in a saccharification step.

SUMMARY

Described are compositions and methods relating to an α-amylase fromBacillus subtilis (AmyE) and related polypeptides. AmyE α-amylase isunique in that it exhibits high specific activity below pH 5.0, and evenat about pH 4-4.5. Furthermore, Ca²⁺ does not affect the thermalstability of AmyE, avoiding the need to add exogenous Ca²⁺ to starchliquefaction or saccharification reactions. These features of AmyEpolypeptides allow liquefaction and saccharification to be performed inthe same reaction mixture (and optionally in the same reaction vessel)without the need to adjust the pH of the reaction mixture betweenliquefaction and saccharification. In particular, reaction conditions donot have to be adjusted when using AmyE and a glucoamylase, avoiding astep and time delays between liquefaction and saccharification, alongwith the potential formation of insoluble amylose aggregates. AmyE can,therefore, be used sequentially or simultaneously with a glucoamylase toliquefy and/or saccharify starch, and at a pH and Ca²⁺ concentrationthat are optimal for the glucoamylase. AmyE also exhibits glucoamylaseactivity, reducing or eliminating the need for an additional polypeptidewith glucoamylase activity to perform saccharification.

In one aspect, a method for liquefying and saccharifying starch in astarch conversion process, is provided, comprising contacting a starchsubstrate with an AmyE polypeptide to form a reaction mixture forliquefying and saccharifying the starch substrate in the reactionmixture to produce glucose, wherein the liquefying and saccharifying areperformed in the same reaction mixture without a pH adjustment.

In some embodiments, the saccharifying (i.e., saccharification) isperformed in the absence of an additional polypeptide havingglucoamylase activity. In some embodiments, the liquefying (i.e.,liquefaction) is performed at a pH suitable for the activity of aglucoamylase polypeptide. In some embodiments, the pH is 5.0 or lower.In some embodiments, the pH is 4.5 or lower. In particular embodiments,the pH is 4.0 or lower. In some embodiments, exogenous calcium is notadded to the reaction mixture. In some embodiments, the calciumconcentration in the reaction mixture is less than about 8 ppm.

In some embodiments, an additional polypeptide having glucoamylaseactivity is added to the reaction mixture before contacting the starchsubstrate with the AmyE polypeptide. In some embodiments, the additionalpolypeptide having glucoamylase activity is added to the reactionmixture after contacting the starch substrate with the AmyE polypeptide.In some embodiments, the additional polypeptide having glucoamylaseactivity is added to the reaction mixture simultaneously with contactingthe starch substrate with the AmyE polypeptide.

In some embodiments, the method further comprises fermenting the glucoseproduced by the liquefying and saccharifying to produce a biofuel, suchas an alcohol. In some embodiments, the alcohol is ethanol. In someembodiments, the alcohol is butanol. In some embodiments, at least aportion of the saccharifying and fermenting occur in the same reactionmixture simultaneously, as in the case of SSF.

In some embodiments, a batch fermentation process is used in a closedsystem, wherein the composition of the reacture mixture (including thepH) is selected at the beginning of the fermentation and is not alteredduring the fermentation. In another embodiment, a “fed-batchfermentation” system is used, wherein the starch substrate is added inincrements as the fermentation progresses. In yet another embodiment, acontinuous fermentation system is used, where a defined fermentationmedium is added continuously to a bioreactor, and an equal amount ofconditioned reaction mixture is removed for processing.

In some embodiments, the saccharified starch solution is converted tofructose-starch based syrup (HFSS), such as HFCS. The conversion to HFSSmay be catalyzed at a pH of about 6.0 to about 8.0, e.g., pH 7.5, andthe product may contain about 40-45% fructose. In some embodiments, themethod further comprises contacting the glucose produced by theliquefying and saccharifying with a glucose isomerase to producefructose (e.g., in the form of HFCS). In some embodiments, exogenouscalcium is not added to the reaction mixture. In some embodiments, thecalcium concentration in the reaction mixture is less than about 8 ppm.In some embodiments, the method further includes contacting the glucoseproduced by the liquefying and saccharifying with a glucose isomerase toproduce fructose without reducing the amount of calcium in the reactionmixture.

In some embodiments, AmyE polypeptides are added to the reaction mixturein an amount of about 0.03-1 kg per metric ton of dry solids (ds). Insome embodiments, the reaction mixture is a starch slurry having about20-35% ds (w/w). The saccharification reaction may be performed at atemperature of about 60° C. to about 90° C., e.g., 70° C. to 85° C., oreven 10, 12, 14, 16, 18, or even 20° C. below the starch gelationtemperature (i.e., about 75° C.), and a pH of about 4.0 to about 6.0,e.g., about 4.2 to about 4.8. in some embodiments, the product of thesaccharification reaction is a glucose-rich syrup. The glucoseconcentration may reach at least about 95% w/w ds.

In some embodiments, the AmyE polypeptides is any naturally-occurringAmyE polypeptide, for example, the AmyE polypeptides having the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequencewith at least about 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%,99%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3, e.g., asmeasured with the BLAST sequence alignment algorithm. In particularembodiments, the AmyE polypeptide used in the method has at least 80%amino acid sequence identity with the amino acid sequence of SEQ IDNO: 1. In particular embodiments, the AmyE polypeptide used in themethod has at least 90% amino acid sequence identity with the amino acidsequence of SEQ ID NO: 1.

In some embodiments, the AmyE polypeptide used in the method includes adeletion of the C-terminal starch binding domain. In particularembodiments, the AmyE polypeptides having the C-terminal deletion hasthe amino acid sequence of SEQ ID NO: 2, or an amino acid sequence withat least about 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%,sequence identity to SEQ ID NO: 2. In particular embodiments, the AmyEpolypeptide is truncated from residue D425 (referring to SEQ ID NO: 1).

In another aspect, AmyE variants having advantageous properties areprovided. The AmyE variants may have an altered property or properties,compared to a wild-type AmyE polypeptide, for example an alteredproperty with respect to specific activity towards starch,maltoheptaose, and/or maltotriose substrates, substrate specificity,thermostability, temperature optima, pH optima, pH and/or temperaturerange, oxidative stability, ability to reduce the viscosity of a starchcomposition, or the like. In some cases, the altered property of theAmyE variant relates to the specific activity on a particular cornflour, maltotriose, maltoheptaose substrate at particular pH (e.g., 4 or5.8), heat stability at a particular temperature, (e.g., 60° C.), orcleaning performance at a particular pH (e.g., 8 or pH 10). The alteredproperty may be characterized by a Performance Index (PI), where the PIis a ratio of performance of the AmyE variant compared to a wild-typeAmyE. In some embodiments, the PI is greater than about 0.5, while inother embodiments, the PI is about 1 or is greater than 1.

In one aspect, the variant polypeptide has α-amylase activity and atleast one altered characteristic that improves enzyme performance, thevariant polypeptide comprising:

an amino acid sequence having at least 60% amino acid sequence identityto a parental α-amylase polypeptide selected from AmyE (SEQ ID NO: 1) ora truncated variant of AmyE (SEQ ID NO: 2), and

a modification at one or more positions selected from the groupconsisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 267, 268, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, and 425.

wherein the modification produces a variant polypeptide having aperformance index (PI) greater than 1.0 for at least one characteristicthat improves enzyme performance.

In some embodiments, the variant polypeptide comprises a modification atone or more positions selected from the group consisting of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 63, 64, 65,66, 67, 68, 69, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 100, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 121, 124,125, 126, 128, 129, 130, 131, 132, 134, 135, 136, 140, 141, 142, 143,144, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162,163, 164, 165, 166, 167, 168, 170, 171, 172, 175, 179, 180, 181, 184,186, 187, 188, 189, 190, 192, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 207, 209, 211, 212, 213, 214, 217, 218, 219, 221, 222,223, 224, 225, 226, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 267, 268,270, 271, 272, 273, 274, 275, 276, 277, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 297, 298, 299,300, 301, 302, 303, 304, 305, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 324, 325, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 355, 356, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, and 425,

wherein the modification produces a variant polypeptide having aperformance index (PI) greater than 0.5 for protein expression, and a PIgreater than 1.1 for at least one characteristic that improves enzymeperformance.

In some embodiments, the one or more positions are selected from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 100, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122,124, 125, 126, 127, 128, 129, 130, 131, 132, 134, 135, 136, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, and 425, which positions are non-fully restrictive forperformance in either the full-length or truncated parental polypeptide.

In some embodiments, the modification is a substitution of one or moreamino residues present in the parental polypeptide to different aminoacid residues, at one or more positions selected from the groupconsisting of 1A, 1C, 1D, 1E, 1F, 1G, 1H, 1K, 1M, 1N, 1Q, 1R, 1S, 1T,1V, 1W, 1Y, 2A, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2K, 2L, 2M, 2N, 2P, 2Q, 2R,2S, 2V, 2W, 2Y, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3K, 3L, 3M, 3N, 3P, 3Q, 3R,3S, 3V, 3W, 3Y, 4C, 4D, 4E, 4F, 4G, 4H, 4I, 4K, 4L, 4M, 4N, 4Q, 4S, 4T,4V, 4W, 4Y, 5A, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5K, 5L, 5N, 5R, 5V, 5W, 5Y,6C, 6D, 6E, 6H, 6K, 6L, 6M, 6N, 6P, 6Q, 6R, 6S, 6T, 6V, 6W, 7A, 7C, 7D,7E, 7F, 7G, 7H, 7I, 7L, 7M, 7N, 7P, 7Q, 7R, 7S, 7T, 7W, 7Y, 8A, 8C, 8E,8F, 8G, 8H, 8I, 8K, 8L, 8M, 8N, 8P, 8Q, 8R, 8T, 8V, 8W, 8Y, 9A, 9C, 9D,9E, 9F, 9H, 9I, 9K, 9M, 9N, 9P, 9R, 9S, 9T, 9V, 9W, 9Y, 10A, 10I, 10L,10M, 10N, 10P, 10Q, 10S, 10y, 11A, 11F, 11G, 11H, 11M, 11S, 11V, 11W,11Y, 12I, 12M, 12V, 13A, 13C, 13D, 13E, 13F, 13G, 13I, 13L, 13M, 13Q,13T, 13V, 13W, 13Y, 14C, 14F, 14G, 14M, 14N, 14S, 14T, 14V, 15A, 15F,16A, 16D, 16E, 16F, 16G, 16H, 16I, 16L, 16M, 16Q, 16S, 16T, 16V, 17A,17F, 17I, 17M, 17Q, 17Y, 18A, 18C, 18D, 18E, 18G, 18H, 18M, 18N, 18Q,18R, 18T, 19A, 19C, 19H, 19L, 19M, 19N, 19S, 19W, 19Y, 20A, 20C, 20D,20F, 20G, 20H, 20I, 20K, 20L, 20M, 20P, 20Q, 20R, 20S, 20T, 20V, 20W,20Y, 21A, 21C, 21D, 21E, 21H, 21I, 21K, 21L, 21M, 21N, 21Q, 21R, 21S,21V, 22I, 22M, 22Q, 22S, 22T, 22V, 23A, 23C, 23D, 23E, 23F, 23G, 23H,23I, 23L, 23M, 23N, 23R, 23S, 23T, 23V, 23W, 23Y, 24A, 24C, 24D, 24F,24G, 24L, 24N, 24P, 24Q, 24R, 24S, 24T, 24V, 24Y, 25A, 25C, 25D, 25E,25F, 25G, 25H, 25I, 25K, 25L, 25R, 25S, 25T, 25V, 25W, 25Y, 26A, 26F,26I, 26L, 26V, 27A, 27C, 27D, 27E, 27F, 27G, 27H, 27I, 27L, 27M, 27N,27P, 27Q, 27R, 27S, 27T, 27V, 27W, 27Y, 28A, 28C, 28F, 28G, 28H, 28I,28K, 28L, 28M, 28N, 28P, 28Q, 28R, 28S, 28T, 28V, 28W, 28Y, 29A, 29C,29F, 29L, 29M, 29T, 29V, 30A, 30C, 30D, 30E, 30F, 30G, 30I, 30K, 30L,30M, 30N, 30P, 30Q, 30R, 30S, 30T, 30V, 30W, 30Y, 31A, 31C, 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272S,272T, 272V, 273D, 273G, 273I, 273K, 273L, 273P, 273Q, 273R, 273S, 273T,273V, 273W, 273Y, 274A, 274C, 274F, 274G, 274H, 274I, 274K, 274L, 274M,274N, 274P, 274Q, 274R, 274S, 274T, 274V, 274W, 274Y, 275A, 275C, 275E,275F, 275G, 275H, 275I, 275K, 275L, 275M, 275N, 275P, 275Q, 275R, 275S,275T, 275V, 275W, 275Y, 276A, 276C, 276D, 276F, 276G, 276H, 276I, 276K,276L, 276M, 276N, 276P, 276Q, 276R, 276S, 276T, 276V, 276W, 276Y, 277A,277C, 277D, 277F, 277G, 277H, 277I, 277K, 277L, 277M, 277N, 277P, 277Q,277R, 277S, 277T, 277V, 277W, 277Y, 278A, 278C, 278T, 279D, 279E, 279G,279H, 279I, 279K, 279L, 279M, 279N, 279P, 279Q, 279R, 279S, 279V, 279W,279Y, 280A, 280D, 280E, 280F, 280G, 280H, 280K, 280L, 280M, 280N, 280Q,280R, 280S, 280T, 280Y, 281C, 281F, 281L, 282A, 282C, 282D, 282E, 282F,282G, 282H, 282I, 282K, 282L, 282M, 282N, 282P, 282Q, 282R, 282T, 282V,282W, 282Y, 283A, 283C, 283F, 283G, 283H, 283I, 283L, 283M, 283N, 283P,283R, 283S, 283T, 283V, 283W, 283Y, 284A, 284C, 284E, 284F, 284G, 284H,284I, 284K, 284L, 284M, 284N, 284P, 284Q, 284R, 284S, 284T, 284V, 284W,284Y, 285A, 285C, 285E, 285H, 285I, 285L, 285M, 285N, 285Q, 285S, 285T,285V, 285Y, 286A, 286C, 286L, 286M, 286N, 286Q, 286T, 286V, 287A, 287C,287D, 287E, 287F, 287G, 287H, 287I, 287K, 287L, 287M, 287N, 287P, 287Q,287S, 287T, 287V, 287W, 287Y, 288A, 288C, 288I, 288M, 288T, 288V, 289A,289S, 290F, 290H, 290M, 290Y, 291C, 291F, 291G, 291I, 291L, 291M, 291N,291S, 291T, 291V, 292A, 292C, 292I, 292L, 292M, 292S, 292T, 292W, 293C,293D, 293E, 293F, 293G, 293N, 293Q, 293S, 293V, 294C, 294G, 294M, 294N,294S, 294T, 294V, 295A, 295C, 295G, 295T, 296A, 296C, 296F, 296G, 296H,296K, 296M, 297A, 297C, 297D, 297E, 297F, 297G, 297H, 297I, 297K, 297L,297M, 297N, 297P, 297Q, 297R, 297T, 297V, 297W, 297Y, 298C, 298D, 298E,298F, 298H, 298I, 298K, 298L, 298M, 298N, 298P, 298Q, 298R, 298S, 298V,298W, 299C, 299D, 299E, 299F, 299G, 299H, 299I, 299L, 299M, 299N, 299P,299Q, 299V, 300A, 300C, 300F, 300H, 300I, 300K, 300L, 300M, 300N, 300Q,300R, 300S, 300V, 300Y, 301C, 301D, 301F, 301H, 301I, 301K, 301L, 301M,301Q, 301R, 301T, 301V, 302C, 302E, 302F, 302G, 302K, 302M, 302N, 302S,302T, 303L, 303M, 303W, 303Y, 304C, 304E, 304G, 304L, 304N, 304Y, 305A,305G, 305I, 305N, 305T, 305V, 307A, 307C, 307D, 307N, 307Q, 307T, 307V,307Y, 308A, 308C, 308D, 308F, 308G, 308H, 308I, 308K, 308L, 308M, 308N,308P, 308Q, 308R, 308S, 308T, 308V, 308W, 308Y, 309C, 309D, 309E, 309F,309H, 309I, 309K, 309M, 309N, 309P, 309R, 309S, 309T, 309V, 309Y, 310A,310D, 310E, 310F, 310H, 310I, 310L, 310M, 310N, 310P, 310Q, 310R, 310S,310T, 310Y, 311A, 311C, 311D, 311E, 311F, 311H, 311K, 311L, 311M, 311N,311P, 311Q, 311R, 311S, 311T, 311V, 311W, 311Y, 312A, 312C, 312D, 312E,312F, 312G, 312H, 312I, 312K, 312L, 312M, 312P, 312Q, 312R, 312S, 312T,312V, 312W, 312Y, 313A, 313C, 313D, 313E, 313F, 313H, 313I, 313K, 313L,313M, 313N, 313P, 313Q, 313R, 313S, 313T, 313V, 313W, 313Y, 314A, 314C,314D, 314F, 314G, 314H, 314K, 314L, 314M, 314Q, 314R, 314S, 314T, 314W,314Y, 315C, 315D, 315E, 315G, 315H, 315I, 315K, 315L, 315M, 315N, 315P,315Q, 315T, 315V, 316C, 316D, 316H, 316I, 316L, 316M, 316Y, 317A, 317C,317D, 317E, 317F, 317G, 317H, 317I, 317K, 317L, 317N, 317Q, 317R, 317S,317T, 317V, 317W, 317Y, 318D, 318F, 318H, 318I, 318K, 318L, 318M, 318N,318R, 318S, 318T, 318V, 318W, 318Y, 319A, 319D, 319F, 319G, 319H, 319L,319N, 319P, 319Q, 319S, 319V, 319W, 320A, 320C, 320D, 320F, 320G, 320H,320I, 320K, 320L, 320M, 320N, 320P, 320Q, 320T, 320V, 320W, 320Y, 321A,321C, 321D, 321E, 321F, 321G, 321H, 321I, 321K, 321L, 321M, 321N, 321P,321R, 321S, 321T, 321V, 321W, 322L, 322M, 322V, 323A, 323C, 323H, 323N,323R, 323S, 323T, 324A, 324C, 324E, 324F, 324G, 324H, 324I, 324K, 324L,324M, 324N, 324P, 324Q, 324R, 324S, 324T, 324V, 324W, 324Y, 325A, 325C,325D, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325N, 325P, 325T, 325V,325W, 325Y, 326A, 326Q, 327C, 327D, 327F, 327G, 327H, 327K, 327N, 327P,327R, 327T, 327V, 327Y, 328C, 328D, 328E, 328F, 328G, 328H, 328I, 328K,328L, 328N, 328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329A, 329D,329E, 329F, 329G, 329H, 329N, 329Q, 329R, 329S, 329T, 330A, 330C, 330H,330L, 330M, 330S, 330W, 330Y, 331C, 331D, 331F, 331G, 331I, 331K, 331L,331M, 331N, 331Q, 331R, 331S, 331T, 331V, 331Y, 332A, 332C, 332E, 332F,332G, 332I, 332K, 332L, 332M, 332Q, 332R, 332S, 332V, 332Y, 333C, 333D,333F, 333G, 333H, 333I, 333K, 333L, 333M, 333N, 333P, 333R, 333S, 333T,333V, 333W, 333Y, 334C, 334D, 334F, 334G, 334H, 334I, 334L, 334M, 334N,334Q, 334R, 334S, 334T, 334V, 334Y, 335A, 335L, 335M, 335Q, 335T, 335V,336A, 336C, 336E, 336F, 336G, 336H, 336I, 336K, 336L, 336M, 336N, 336Q,336R, 336S, 336V, 336W, 336Y, 337D, 337G, 337H, 337K, 337L, 337N, 337P,337Q, 337R, 337S, 337V, 337W, 337Y, 338C, 338F, 338G, 338I, 338L, 338M,338N, 338P, 338S, 338T, 339C, 339G, 339I, 339S, 339T, 339V, 340D, 340E,340F, 340G, 340H, 340I, 340K, 340L, 340M, 340N, 340S, 340T, 340V, 340W,341A, 341I, 341L, 341M, 341V, 341W, 341Y, 342A, 342D, 342E, 342F, 342G,342K, 342L, 342M, 342N, 342R, 342S, 342V, 342Y, 343A, 343C, 343D, 343E,343F, 343G, 343H, 343I, 343K, 343L, 343M, 343P, 343Q, 343S, 343T, 343V,343W, 343Y, 344A, 344C, 344D, 344E, 344F, 344G, 344H, 344I, 344K, 344L,344M, 344N, 344Q, 344R, 344S, 344T, 344W, 344Y, 345A, 345C, 345D, 345E,345F, 345G, 345H, 345I, 345N, 345Q, 345S, 345T, 345V, 345W, 345Y, 346C,346D, 346E, 346F, 346H, 346I, 346K, 346L, 346M, 346N, 346P, 346R, 346S,346T, 346V, 346Y, 347A, 347C, 347D, 347E, 347F, 347H, 347I, 347K, 347L,347M, 347N, 347P, 347Q, 347R, 347S, 347T, 347V, 347W, 347Y, 348C, 348F,348G, 348H, 348I, 348K, 348M, 348N, 348P, 348R, 348S, 348T, 348V, 348W,348Y, 349A, 349C, 349D, 349F, 349G, 349H, 349I, 349K, 349L, 349M, 349N,349Q, 349R, 349S, 349T, 349V, 349W, 349Y, 350A, 350C, 350D, 350N, 350S,351A, 351D, 351G, 351H, 351K, 351L, 351M, 351P, 351Q, 351R, 351S, 351T,351V, 351W, 351Y, 352A, 352D, 352E, 352F, 352G, 352H, 352I, 352K, 352N,352Q, 352R, 352T, 352V, 352W, 352Y, 353A, 353C, 353D, 353E, 353F, 353G,353I, 353K, 353L, 353M, 353N, 353Q, 353R, 353T, 353V, 353W, 353Y, 354A,354C, 354M, 354P, 354Q, 354S, 354T, 355C, 355D, 355E, 355F, 355G, 355I,355K, 355L, 355M, 355N, 355T, 355V, 355W, 355Y, 356D, 356E, 356F, 356G,356H, 356I, 356K, 356L, 356M, 356P, 356Q, 356T, 356W, 356Y, 357A, 357C,357D, 357E, 357F, 357H, 357I, 357K, 357L, 357M, 357N, 357P, 357Q, 357R,357S, 357T, 357V, 357W, 357Y, 358A, 358C, 358D, 358E, 358F, 358G, 358H,358I, 358K, 358L, 358M, 358P, 358Q, 358R, 358S, 358T, 358V, 358W, 358Y,359A, 359C, 359D, 359E, 359F, 359G, 359H, 359I, 359K, 359L, 359M, 359P,359Q, 359R, 359S, 359T, 359V, 359W, 359Y, 360F, 360H, 360L, 360N, 360P,360R, 360T, 360W, 361A, 361C, 361G, 361H, 361L, 361M, 361N, 361Q, 361S,361T, 361V, 361W, 361Y, 362A, 362C, 362E, 362H, 362I, 362L, 362M, 362Q,362S, 362T, 362V, 362Y, 363D, 363E, 363F, 363G, 363H, 363N, 363Q, 363R,363S, 363V, 363W, 363Y, 364A, 364C, 364D, 364E, 364G, 364I, 364L, 364M,364Q, 364S, 364T, 364V, 365A, 365C, 365D, 365F, 365G, 365I, 365K, 365L,365M, 365N, 365R, 365S, 365T, 365V, 365W, 365Y, 366A, 366C, 366E, 366F,366G, 366H, 366K, 366L, 366M, 366S, 366T, 366V, 367A, 367C, 367D, 367E,367F, 367H, 367I, 367K, 367L, 367M, 367N, 367P, 367R, 367S, 367T, 367V,367W, 367Y, 368D, 368F, 368G, 368I, 368K, 368L, 368M, 368N, 368P, 368Q,368R, 368T, 368V, 368W, 368Y, 369A, 369C, 369D, 369E, 369F, 369G, 369I,369K, 369L, 369M, 369N, 369P, 369Q, 369R, 369S, 369T, 369V, 369W, 369Y,370A, 371A, 371C, 371F, 371G, 371H, 371I, 371L, 371M, 371N, 371Q, 371S,371T, 371W, 371Y, 372A, 372C, 372G, 372I, 372L, 372M, 372N, 372Q, 372S,372T, 373A, 373C, 373F, 373G, 373I, 373M, 373Q, 373S, 373T, 373V, 373W,373Y, 374C, 374E, 374G, 374I, 374L, 374M, 374N, 374S, 374T, 374V, 375A,375C, 375D, 375F, 375G, 375H, 375L, 375M, 375Q, 375S, 375T, 375V, 375W,375Y, 376C, 376D, 376E, 376F, 376G, 376H, 376I, 376L, 376M, 376N, 376P,376Q, 376S, 376T, 376V, 377F, 377H, 377I, 377K, 377L, 377P, 377T, 377W,377Y, 378A, 378C, 378D, 378E, 378F, 378G, 378H, 378I, 378K, 378L, 378M,378N, 378P, 378Q, 378R, 378T, 378V, 378W, 378Y, 379A, 379D, 379G, 379H,379I, 379K, 379L, 379Q, 379T, 379W, 379Y, 380A, 380C, 380D, 380E, 380F,380G, 380H, 380I, 380L, 380M, 380N, 380P, 380Q, 380R, 380T, 380V, 380W,380Y, 381A, 381G, 381I, 381K, 381N, 381P, 381Q, 381R, 381S, 381T, 381W,381Y, 382A, 382C, 382D, 382F, 382G, 382H, 382I, 382K, 382L, 382M, 382N,382P, 382Q, 382R, 382T, 382V, 382W, 382Y, 383A, 383C, 383E, 383F, 383G,383H, 383L, 383N, 383P, 383Q, 383S, 383T, 383V, 383W, 383Y, 384A, 384D,384F, 384G, 384H, 384I, 384K, 384L, 384P, 384Q, 384S, 384T, 384V, 384W,385A, 385C, 385D, 385E, 385F, 385G, 385H, 385I, 385K, 385L, 385M, 385N,385P, 385Q, 385R, 385S, 385V, 385W, 385Y, 386C, 386D, 386F, 386G, 386H,386I, 386L, 386N, 386P, 386R, 386S, 386T, 386V, 386W, 386Y, 387A, 387D,387E, 387G, 387I, 387L, 387N, 387Q, 387S, 388A, 388C, 388D, 388E, 388F,388G, 388H, 388I, 388L, 388M, 388N, 388P, 388Q, 388R, 388S, 388T, 388V,388W, 388Y, 389C, 389E, 389F, 389H, 389I, 389K, 389M, 389N, 389Q, 389S,389T, 389V, 389W, 389Y, 390A, 390C, 390D, 390E, 390F, 390G, 390H, 390I,390K, 390L, 390M, 390N, 390R, 390S, 390T, 390V, 390W, 390Y, 391E, 391F,391G, 391H, 391I, 391K, 391L, 391N, 391P, 391R, 391S, 391T, 391V, 391W,391Y, 392A, 392C, 392D, 392E, 392F, 392H, 392K, 392L, 392M, 392N, 392Q,392R, 392S, 392V, 392Y, 393A, 393C, 393D, 393F, 393G, 393H, 393I, 393L,393M, 393P, 393Q, 393S, 393T, 393V, 393W, 393Y, 394A, 394C, 394E, 394F,394H, 394I, 394K, 394L, 394M, 394N, 394P, 394Q, 394R, 394S, 394T, 394V,394W, 395A, 395C, 395E, 395F, 395G, 395H, 395I, 395K, 395L, 395M, 395N,395P, 395Q, 395R, 395S, 395T, 395V, 395W, 395Y, 396A, 396C, 396D, 396E,396G, 396M, 396P, 396S, 396T, 397A, 397C, 397D, 397E, 397F, 397G, 397H,397I, 397L, 397M, 397P, 397R, 397S, 397T, 397V, 397W, 398C, 398D, 398E,398F, 398G, 398I, 398L, 398M, 398N, 398P, 398Q, 398R, 398S, 398T, 398V,398W, 398Y, 399A, 399C, 399D, 399E, 399F, 399H, 399I, 399K, 399L, 399P,399R, 399S, 399T, 399V, 399W, 399Y, 400C, 400D, 400E, 400F, 400G, 400H,400I, 400K, 400L, 400M, 400N, 400P, 400Q, 400R, 400S, 400T, 400V, 400W,400Y, 401A, 401C, 401D, 401E, 401F, 401H, 401I, 401K, 401L, 401M, 401N,401P, 401Q, 401R, 401S, 401T, 401V, 401W, 401Y, 402A, 402C, 402D, 402E,402F, 402G, 402H, 402I, 402K, 402L, 402M, 402N, 402P, 402Q, 402R, 402T,402V, 402W, 402Y, 403A, 403C, 403E, 403G, 403H, 403I, 403M, 403N, 403Q,403S, 403T, 403V, 403W, 403Y, 404D, 404E, 404F, 404G, 404H, 404I, 404L,404M, 404N, 404P, 404R, 404T, 404V, 404W, 404Y, 405E, 405F, 405G, 405H,405I, 405K, 405Q, 405S, 405T, 406D, 406F, 406L, 406T, 406Y, 407A, 407C,407E, 407F, 407G, 407H, 407I, 407K, 407M, 407N, 407P, 407Q, 407R, 407S,407T, 407V, 407W, 407Y, 408A, 408D, 408E, 408F, 408H, 408I, 408K, 408N,408P, 408Q, 408S, 408T, 408V, 408Y, 409A, 409C, 409D, 409E, 409F, 409H,409I, 409L, 409M, 409Q, 409R, 409T, 409V, 409W, 409Y, 410F, 410G, 410I,410K, 410Q, 410S, 410T, 410V, 410W, 410Y, 411A, 411D, 411E, 411F, 411G,411H, 411I, 411L, 411M, 411N, 411Q, 411R, 411S, 411V, 411W, 411Y, 412A,412D, 412E, 412H, 412I, 412K, 412L, 412M, 412N, 412R, 412S, 412T, 412V,412Y, 413C, 413E, 413F, 413G, 413I, 413L, 413M, 413N, 413P, 413R, 413S,413V, 413W, 413Y, 414A, 414C, 414E, 414F, 414G, 414H, 414L, 414M, 414N,414P, 414Q, 414T, 414V, 414W, 415D, 415E, 415F, 415G, 415H, 415I, 415K,415P, 415Q, 415R, 415V, 415W, 416F, 416I, 416L, 416P, 416Q, 416R, 416T,416V, 416Y, 417A, 417C, 417D, 417F, 417G, 417H, 417I, 417K, 417M, 417N,417P, 417Q, 417S, 417W, 417Y, 418C, 418D, 418E, 418F, 418H, 418I, 418K,418N, 418Q, 418R, 418T, 418W, 418Y, 419C, 419D, 419E, 419F, 419G, 419H,419I, 419L, 419P, 419Q, 419S, 419T, 419Y, 420D, 420E, 420F, 420G, 420H,420I, 420K, 420L, 420M, 420N, 420Q, 420R, 420S, 420T, 420V, 420W, 420Y,421A, 421C, 421G, 421I, 421L, 421M, 421S, 421T, 422A, 422F, 422G, 422H,422I, 422M, 422N, 422Q, 422S, 422V, 422W, 422Y, 423A, 423D, 423G, 423H,423I, 423K, 423P, 423Q, 423R, 423T, 423V, 423W, 424D, 424E, 424G, 424I,424K, 424M, 424N, 424Q, 424R, 424S, 424T, 424V, 424W, 424Y, 425A, 425I,425K, 425L, 425M, 425S, 425T, 425V, 425W, and 425Y.

In some embodiments, the modification is a substitution of one or moreamino residues present in the parental polypeptide to different aminoacid residues, at one or more positions selected from the groupconsisting of 1A, 1D, 1F, 1G, 1H, 1K, 1M, 1N, 1Q, 1R, 1S, 1T, 1V, 1W,1Y, 2A, 2E, 2F, 2G, 2H, 2I, 2P, 2Q, 2R, 2S, 2W, 3D, 3E, 3F, 3G, 3H, 3I,3K, 3L, 3M, 3N, 3P, 3Q, 3R, 3S, 3V, 3W, 3Y, 4D, 4E, 4F, 4G, 4I, 4K, 4L,4Q, 4S, 4T, 4V, 4W, 5A, 5D, 5E, 5F, 5G, 5K, 5L, 5V, 5W, 6D, 6H, 6K, 6L,6P, 6Q, 6S, 6V, 6W, 7A, 7D, 7E, 7H, 7N, 7Q, 7R, 7S, 8A, 8C, 8E, 8F, 8G,8H, 8I, 8K, 8L, 8N, 8P, 8Q, 8R, 8T, 8V, 8W, 8Y, 9A, 9D, 9E, 9F, 9H, 9I,9K, 9M, 9N, 9P, 9R, 9V, 9W, 9Y, 10I, 10L, 10M, 10P, 10S, 10y, 11A, 11F,11M, 11V, 12I, 12M, 13A, 13D, 13Q, 14G, 14S, 14T, 14V, 15F, 16M, 16Q,18G, 18N, 18R, 19H, 19W, 20A, 20D, 20F, 20G, 20H, 20I, 20K, 20M, 20R,20S, 20V, 20W, 20Y, 21E, 21I, 21M, 21Q, 21S, 21V, 22I, 22T, 22V, 23A,23D, 23E, 23F, 23G, 23H, 23I, 23L, 23M, 23N, 23R, 23S, 23T, 23V, 23W,23Y, 24A, 24C, 24F, 24G, 24R, 24S, 24T, 24V, 24Y, 25E, 25F, 25K, 25L,25R, 25S, 25T, 25V, 25W, 25Y, 26I, 26L, 27A, 27E, 27F, 27G, 27H, 27I,27L, 27P, 27Q, 27R, 27S, 27T, 27V, 27W, 27Y, 28A, 28C, 28G, 28H, 28I,28K, 28L, 28M, 28N, 28P, 28Q, 28R, 28S, 28V, 28W, 28Y, 29F, 29L, 29V,30A, 30C, 30D, 30E, 30F, 30G, 30L, 30M, 30N, 30Q, 30R, 30S, 30T, 30V,30W, 30Y, 31A, 31F, 31G, 31H, 31I, 31K, 31L, 31M, 31N, 31Q, 31S, 31T,31V, 31Y, 32G, 32S, 33A, 33D, 33E, 33H, 33Q, 33S, 34W, 35A, 35F, 35G,35H, 35I, 35L, 35M, 35N, 35Q, 35R, 35S, 35V, 35W, 36F, 36H, 36I, 36L,36S, 36T, 36Y, 37L, 37V, 39A, 39P, 39S, 39V, 42V, 43A, 43S, 43T, 43V,44A, 44D, 44E, 44F, 44G, 44H, 44I, 44K, 44N, 44R, 44S, 44T, 44Y, 45F,45H, 45I, 45L, 45M, 45S, 45T, 46A, 46D, 46F, 46H, 46L, 46M, 46N, 46R,47A, 47D, 47F, 47G, 47H, 47I, 47K, 47L, 47N, 47P, 47R, 47S, 47T, 47V,47Y, 48A, 48E, 48F, 48H, 48N, 48P, 48W, 49A, 49F, 49G, 49H, 49K, 49L,49Q, 49R, 49S, 49T, 49V, 49W, 49Y, 50E, 50F, 50H, 50I, 50K, 50L, 50M,50P, 50R, 50S, 50T, 50V, 50W, 50Y, 51D, 51E, 51F, 51H, 51I, 51K, 51L,51P, 51Q, 51R, 51S, 51T, 51V, 51W, 52E, 52F, 52G, 52H, 52I, 52K, 52L,52M, 52N, 52Q, 52R, 52S, 52T, 52V, 52W, 52Y, 53A, 53E, 53F, 53H, 53I,53L, 53P, 53R, 53S, 53T, 53V, 54A, 54C, 54F, 54G, 54H, 54L, 54N, 54P,54R, 54T, 54W, 54Y, 55A, 55F, 55H, 55N, 55P, 55Q, 55S, 55T, 55Y, 56D,56E, 56F, 56G, 56I, 56K, 56L, 56P, 56Q, 56R, 56T, 56V, 56W, 56Y, 57A,57E, 57H, 57M, 57Q, 57R, 57S, 57Y, 58F, 59A, 59C, 59F, 59H, 59N, 59P,59R, 59S, 59T, 59W, 60L, 60N, 63H, 63N, 64A, 64S, 65A, 65I, 65R, 66D,66E, 66G, 66M, 66N, 66Q, 66R, 67A, 67F, 67G, 67I, 67L, 67N, 67Q, 67T,67W, 68D, 68F, 68H, 68I, 68L, 68N, 68R, 68S, 68T, 68V, 68W, 69M, 69V,72E, 72F, 72G, 72H, 72I, 72K, 72Q, 72S, 72T, 72V, 72W, 72Y, 73F, 73M,73W, 74M, 74T, 76A, 76L, 76M, 76P, 76Q, 76R, 76Y, 77A, 77D, 77K, 77L,77R, 77Y, 78D, 78E, 78F, 78G, 78H, 78I, 78K, 78L, 78P, 78R, 78S, 78T,78W, 78Y, 79A, 79M, 79Q, 79S, 80M, 81E, 81G, 81H, 81L, 81M, 81N, 81Q,81R, 81S, 81T, 81V, 81Y, 82D, 82F, 82G, 82I, 82K, 82L, 82M, 82Q, 82R,82S, 82T, 82Y, 83A, 83F, 83L, 84A, 84N, 84S, 84T, 85D, 85E, 85F, 85G,85I, 85K, 85R, 85S, 85T, 85V, 85W, 86D, 86E, 86F, 86G, 86I, 86K, 86L,86M, 86N, 86Q, 86R, 86S, 86V, 86W, 86Y, 87G, 88A, 88D, 88F, 88G, 88H,88K, 88L, 88M, 88N, 88Q, 88R, 88S, 88T, 88W, 88Y, 89D, 89F, 89G, 89H,89I, 89K, 89L, 89M, 89N, 89P, 89Q, 89R, 89S, 89T, 89V, 89W, 89Y, 90D,90E, 90F, 90H, 90I, 90K, 90M, 90N, 90R, 90S, 90T, 90V, 90W, 91D, 91E,91H, 91K, 91N, 91Q, 91R, 91S, 92L, 92V, 93A, 93D, 93G, 93M, 93N, 93R,93S, 93Y, 94I, 95F, 95M, 96I, 98C, 99I, 100C, 100F, 100M, 100V, 103A,103C, 103V, 104A, 104S, 105C, 105D, 105E, 105F, 105G, 105M, 105W, 105Y,106E, 106H, 106N, 106Q, 106S, 106T, 106Y, 107A, 107C, 107E, 107F, 107G,107H, 107I, 107K, 107L, 107M, 107N, 107P, 107Q, 107R, 107S, 107T, 107V,107W, 108C, 108D, 108E, 108F, 108G, 108H, 108I, 108K, 108L, 108N, 108P,108R, 108S, 108T, 108V, 108W, 108Y, 109D, 109H, 109I, 109K, 109L, 109N,109R, 109S, 109V, 109W, 109Y, 110V, 111C, 111E, 111F, 111G, 111H, 111K,111L, 111M, 111N, 111Q, 111R, 111T, 112A, 112D, 112E, 112H, 112K, 112L,112R, 112S, 112T, 112W, 112Y, 113A, 114L, 115A, 115H, 115I, 115L, 115R,115V, 115Y, 116A, 116F, 116G, 116H, 116I, 116L, 116N, 116Q, 116R, 116T,116V, 116W, 116Y, 118D, 118F, 118G, 118H, 118K, 118L, 118M, 118N, 118Q,118R, 118S, 118T, 118V, 118W, 118Y, 119E, 119F, 119I, 119K, 119L, 119M,119Q, 119S, 119T, 119Y, 121S, 124A, 124K, 124Q, 124R, 124S, 124T, 125A,125D, 125F, 125I, 125K, 125Q, 125R, 125V, 125Y, 126A, 126C, 126D, 126F,126G, 126H, 126I, 126K, 126L, 126N, 126P, 126R, 126S, 126T, 126V, 126W,126Y, 128A, 128C, 128E, 128F, 128G, 128H, 128I, 128L, 128M, 128N, 128Q,128R, 128S, 128T, 128V, 129C, 129D, 129E, 130A, 130F, 130L, 130T, 130Y,131A, 131C, 131D, 131F, 131G, 131H, 131I, 131K, 131L, 131N, 131Q, 131T,131V, 131W, 131Y, 132I, 132N, 132S, 132W, 134E, 134F, 134L, 134M, 134R,134Y, 135E, 136L, 140A, 141F, 141H, 142C, 142D, 142F, 142G, 142H, 142I,142K, 142M, 142Q, 142R, 142S, 142T, 142W, 142Y, 143C, 143D, 143K, 143L,143N, 143Q, 143S, 144T, 147F, 147L, 150H, 151C, 151D, 151E, 151G, 151H,151K, 151L, 151M, 151Q, 151S, 151T, 152A, 152C, 152E, 152F, 152G, 152H,152I, 152K, 152L, 152M, 152N, 152P, 152Q, 152R, 152S, 152V, 152W, 152Y,153E, 153F, 153H, 153K, 153L, 153N, 153R, 153T, 153V, 153W, 153Y, 154A,155M, 156A, 156F, 156G, 156K, 156L, 156Q, 156R, 156V, 156Y, 157F, 157H,158A, 158I, 158M, 158T, 158V, 159H, 159I, 159L, 159M, 160A, 160C, 160D,160E, 160F, 160G, 160H, 160I, 160K, 160L, 160M, 160Q, 160S, 160T, 160V,162I, 162M, 163A, 163E, 163F, 163G, 163H, 163I, 163K, 163L, 163N, 163Q,163R, 163S, 163T, 163V, 163W, 163Y, 164G, 164H, 164L, 164N, 164S, 164T,164V, 164W, 164Y, 165C, 165I, 165L, 165M, 165T, 165V, 166C, 166I, 166M,166V, 167A, 167C, 167E, 167F, 167G, 167I, 167K, 167L, 167M, 167Q, 167R,167S, 167T, 167V, 167W, 167Y, 168C, 168E, 168F, 168G, 168K, 168L, 168M,168N, 168S, 168T, 168V, 168W, 168Y, 170C, 171E, 171H, 171I, 171M, 171N,171Q, 171R, 172A, 175Y, 179A, 179C, 179G, 179H, 179S, 179W, 180M, 181V,184D, 186E, 187E, 187F, 187H, 187I, 187K, 187M, 187Q, 187S, 187V, 187W,188A, 188D, 188F, 188G, 188I, 188K, 188L, 188M, 188P, 188Q, 188R, 188T,188V, 189F, 189W, 190H, 190K, 190Q, 190R, 190S, 192G, 192K, 192L, 192P,192S, 192V, 195D, 195F, 195G, 195H, 195K, 195M, 195R, 195V, 195W, 196A,196C, 196E, 196F, 196H, 196I, 196K, 196L, 196M, 196Q, 196R, 196S, 196T,196V, 196Y, 197L, 197V, 198A, 198C, 198I, 198L, 198V, 199C, 199D, 199E,199F, 199H, 199R, 199S, 199T, 199Y, 200I, 200N, 200S, 200V, 201C, 201D,201E, 201F, 201G, 201H, 201I, 201K, 201L, 201N, 201Q, 201R, 201T, 201V,201W, 201Y, 202C, 202V, 203A, 203C, 203F, 203G, 203I, 203K, 203L, 203Q,203R, 203S, 203T, 203V, 203W, 203Y, 204I, 204M, 204W, 204Y, 205A, 205C,205I, 205L, 205M, 205N, 205V, 207A, 209L, 209V, 211H, 211S, 211T, 212G,212N, 213A, 213E, 213F, 213G, 213I, 213K, 213L, 213M, 213P, 213Q, 213R,213T, 213V, 214C, 214D, 214F, 214G, 214I, 214K, 214L, 214M, 214N, 214Q,214R, 214S, 214T, 214V, 214W, 214Y, 217I, 217Q, 217T, 218C, 218D, 218E,218F, 218G, 218H, 218I, 218K, 218L, 218M, 218P, 218Q, 218R, 218S, 218T,218V, 218W, 218Y, 219D, 219F, 219G, 219H, 219I, 219N, 219Q, 219S, 219T,219V, 219Y, 221C, 221E, 221G, 221Q, 221S, 221V, 222F, 222T, 223H, 223L,223M, 223W, 224I, 225E, 225F, 225N, 225P, 225Q, 225T, 225Y, 226I, 226L,229D, 229E, 229N, 229T, 230A, 230D, 230E, 230F, 230H, 230I, 230K, 230M,230Q, 230R, 230S, 230V, 230Y, 231H, 231W, 232S, 233A, 233D, 233E, 233F,233G, 233I, 233K, 233L, 233M, 233N, 233Q, 233S, 233T, 233V, 233W, 233Y,234A, 234F, 234G, 234H, 234I, 234L, 234M, 234N, 234Q, 234R, 234T, 234V,234W, 234Y, 235L, 235M, 236A, 236G, 236I, 236L, 236M, 236N, 236Q, 237C,237D, 237E, 237F, 237G, 237H, 237I, 237K, 237L, 237R, 237T, 237V, 237W,237Y, 238C, 238E, 238G, 238N, 238R, 238S, 238W, 239I, 239M, 240A, 240E,240F, 240G, 240L, 240Q, 240R, 240T, 240V, 240Y, 241F, 241G, 241H, 241I,241K, 241L, 241R, 241S, 241T, 241V, 241W, 241Y, 242A, 242C, 242D, 242F,242I, 242K, 242L, 242S, 242T, 242V, 242W, 242Y, 243D, 243E, 243F, 243G,243H, 243I, 243K, 243L, 243M, 243Q, 243R, 243S, 243T, 243V, 243W, 243Y,244I, 244M, 244V, 245C, 245F, 245H, 245I, 245L, 245M, 245N, 245P, 245R,245T, 245V, 245W, 245Y, 246C, 246D, 246E, 246G, 246I, 246L, 246Q, 246W,246Y, 247F, 247G, 247H, 247I, 247L, 247M, 247N, 247Q, 247T, 247V, 247Y,248F, 248G, 248K, 248L, 248Q, 248R, 248S, 248T, 248V, 248W, 249A, 249C,249F, 249L, 249M, 249V, 250C, 250E, 250F, 250G, 250H, 250I, 250K, 250L,250M, 250T, 250V, 250W, 250Y, 251A, 251C, 251D, 251E, 251G, 251K, 251L,251M, 251P, 251Q, 251V, 251Y, 252F, 252L, 252W, 253F, 253I, 253K, 253L,253M, 253R, 253T, 253W, 253Y, 254A, 254F, 254G, 254H, 254I, 254L, 254N,254T, 254V, 254Y, 255A, 255E, 255I, 255K, 255P, 255R, 255S, 255V, 256A,256C, 256I, 257E, 257I, 257L, 257P, 258C, 258D, 258E, 258N, 258Q, 258R,258S, 258V, 259A, 259G, 259H, 259K, 259Q, 259R, 259S, 259T, 259W, 260A,260C, 260D, 260F, 260H, 260N, 260Q, 260R, 260S, 260Y, 261M, 262I, 263C,263L, 263M, 263S, 263V, 264E, 264H, 264I, 264L, 264Y, 267A, 267C, 267N,267T, 268M, 268Q, 270F, 270G, 270N, 270S, 270V, 271F, 272G, 272L, 272S,272V, 273G, 273I, 273L, 273T, 273Y, 274F, 274G, 274H, 274I, 274K, 274L,274M, 274N, 274P, 274Q, 274R, 274S, 274T, 274V, 274W, 274Y, 275F, 275G,275H, 275K, 275P, 275Q, 275R, 275S, 275T, 275V, 276A, 276C, 276D, 276F,276G, 276H, 276I, 276K, 276L, 276M, 276N, 276P, 276Q, 276R, 276S, 276T,276Y, 277A, 277D, 277F, 277G, 277H, 277I, 277K, 277L, 277N, 277P, 277Q,277R, 277S, 277T, 277V, 277Y, 279H, 279K, 279L, 279M, 279N, 279Q, 279Y,280F, 280Y, 281C, 281L, 282A, 282D, 282I, 282K, 282L, 282M, 282N, 282Q,282T, 282W, 282Y, 283C, 283G, 283H, 283P, 283R, 283S, 283T, 283V, 283W,284A, 284C, 284E, 284F, 284G, 284H, 284I, 284K, 284L, 284N, 284R, 284S,284T, 284V, 284W, 284Y, 285E, 285M, 286C, 286L, 286M, 286V, 287A, 287C,287E, 287H, 287I, 287K, 287L, 287M, 287Q, 287S, 287T, 287V, 288C, 288I,288M, 288V, 289A, 290Y, 291C, 291G, 291L, 291S, 291T, 292A, 292C, 292I,292L, 292T, 293C, 293V, 294C, 294G, 294S, 294T, 295A, 295G, 297D, 297E,297F, 297G, 297H, 297I, 297K, 297L, 297M, 297N, 297P, 297Q, 297R, 297T,297V, 297W, 298C, 298D, 298E, 298F, 298H, 298I, 298K, 298L, 298M, 298N,298P, 298Q, 298R, 298S, 298V, 298W, 299C, 299G, 299I, 299N, 299V, 300H,300M, 300R, 300V, 301I, 301K, 301L, 301M, 301T, 302T, 303M, 304L, 304Y,305T, 305V, 307C, 307N, 308C, 308F, 308G, 308H, 308I, 308K, 308L, 308M,308N, 308P, 308Q, 308R, 308S, 308T, 308V, 308W, 308Y, 309D, 309E, 309F,309H, 309K, 309R, 309S, 310A, 311A, 311H, 311K, 311R, 312D, 312F, 312G,312H, 312I, 312K, 312L, 312M, 312P, 312Q, 312R, 312S, 312T, 312V, 312W,312Y, 313A, 313D, 313E, 313F, 313K, 313L, 313N, 313Q, 313R, 313S, 313W,313Y, 314A, 314F, 314H, 314K, 314L, 314M, 314Q, 314R, 314S, 314T, 314W,314Y, 315K, 315N, 315P, 315T, 316Y, 317A, 317C, 317E, 317F, 317H, 317K,317L, 317R, 317S, 317T, 317V, 317W, 317Y, 318D, 318F, 318H, 318I, 318K,318L, 318M, 318N, 318R, 318S, 318T, 318V, 318W, 318Y, 319G, 319L, 319N,319Q, 319V, 319W, 320C, 320F, 320G, 320I, 320K, 320L, 320M, 320P, 320Q,320T, 320V, 320Y, 321C, 321D, 321E, 321F, 321G, 321H, 321I, 321K, 321L,321R, 321S, 321T, 321V, 321W, 322L, 322M, 322V, 324A, 324F, 324G, 324H,324I, 324K, 324L, 324M, 324N, 324Q, 324R, 324S, 324T, 324V, 324W, 324Y,325C, 325D, 325G, 325H, 325I, 325K, 325L, 325M, 325N, 325P, 325T, 325V,327C, 327D, 327G, 327H, 327N, 327T, 328D, 328E, 328F, 328L, 328N, 328Q,328Y, 329F, 329H, 329Q, 330W, 330Y, 331D, 331F, 331G, 331I, 331L, 331Q,331S, 331T, 331V, 331Y, 332A, 332C, 332G, 332Q, 332S, 333C, 333G, 333H,333K, 333L, 333M, 333R, 333S, 333W, 333Y, 334D, 334H, 334I, 334L, 334M,334N, 334R, 334T, 335V, 336A, 336C, 336F, 336G, 336I, 336M, 336N, 336Q,336R, 336V, 336W, 336Y, 337H, 337N, 337S, 337V, 337W, 337Y, 338G, 338I,338L, 338M, 338S, 338T, 339C, 340F, 340H, 340K, 340L, 340M, 340N, 340S,340T, 340V, 340W, 341A, 341L, 341Y, 342A, 342K, 342N, 342R, 342Y, 343A,343D, 343E, 343F, 343H, 343K, 343L, 343M, 343Q, 343S, 343T, 343W, 343Y,344A, 344D, 344E, 344F, 344G, 344I, 344K, 344L, 344M, 344N, 344Q, 344R,344S, 344T, 344W, 344Y, 345C, 345E, 345F, 345G, 345H, 345I, 345N, 345Q,345S, 345T, 345V, 346C, 346D, 346E, 346I, 346K, 346L, 346M, 346N, 346S,346T, 346V, 346Y, 347D, 347F, 347H, 347I, 347K, 347L, 347M, 347Q, 347R,347S, 347T, 347V, 347W, 348F, 348H, 348I, 348K, 348R, 348S, 348T, 348V,348W, 348Y, 349A, 349F, 349G, 349I, 349K, 349M, 349N, 349R, 349S, 349V,349W, 349Y, 350D, 351A, 351D, 351G, 351H, 351K, 351L, 351M, 351P, 351Q,351R, 351T, 351V, 351W, 351Y, 352A, 352H, 352Q, 352T, 352Y, 353A, 353D,353E, 353G, 353I, 353K, 353L, 353M, 353Q, 353V, 353W, 353Y, 355C, 355F,355I, 355L, 355M, 355V, 355Y, 356D, 356F, 356G, 356I, 356K, 356L, 356P,356Q, 356T, 356W, 356Y, 357A, 357H, 357I, 357K, 357L, 357N, 357Q, 357R,357S, 357T, 357V, 357W, 357Y, 358C, 358D, 358F, 358G, 358H, 358I, 358K,358L, 358M, 358Q, 358R, 358S, 358T, 358V, 358Y, 359D, 359E, 359H, 359L,359M, 359P, 359Q, 359R, 359T, 359V, 359W, 360F, 360P, 360T, 361C, 361L,361M, 361N, 361Q, 361S, 361T, 361V, 362A, 362C, 362I, 362L, 362V, 362Y,363D, 363G, 363H, 363Q, 363R, 363S, 363V, 363W, 363Y, 364A, 364C, 364G,364I, 364L, 364M, 364Q, 364S, 364T, 364V, 365C, 365I, 365K, 365L, 365N,365R, 365S, 365V, 366A, 366K, 367L, 367M, 367N, 367R, 367S, 367T, 367W,367Y, 368G, 368I, 368K, 368L, 368R, 368T, 368V, 368W, 369C, 369D, 369E,369F, 369G, 369I, 369K, 369L, 369N, 369Q, 369S, 369T, 369V, 369Y, 371A,371C, 371F, 371I, 371L, 371M, 371N, 371S, 371T, 371Y, 372A, 372C, 372I,372L, 372N, 372S, 372T, 373A, 373C, 373F, 373I, 373M, 373T, 373V, 374C,374G, 374I, 374M, 374S, 374T, 374V, 375A, 375C, 375D, 375F, 375H, 375L,375M, 375Q, 375S, 375T, 375Y, 376G, 376I, 376S, 376T, 376V, 377F, 377H,377L, 377T, 377W, 377Y, 378C, 378E, 378F, 378G, 378H, 378I, 378K, 378L,378M, 378N, 378Q, 378R, 378T, 378V, 378W, 378Y, 379A, 379G, 379H, 379I,379K, 379L, 379Q, 379T, 379Y, 380C, 380E, 380F, 380G, 380H, 380L, 380M,380N, 380P, 380Q, 380R, 380T, 380V, 380W, 380Y, 381G, 381I, 381Q, 381R,381S, 381T, 381W, 381Y, 382A, 382C, 382F, 382I, 382K, 382Q, 382R, 382T,382W, 382Y, 383A, 383F, 383L, 383P, 383Q, 383V, 384A, 384G, 384H, 384I,384K, 384P, 384Q, 384V, 384W, 385C, 385F, 385H, 385I, 385K, 385L, 385N,385P, 385Q, 385R, 385S, 385V, 385W, 385Y, 386D, 386F, 386G, 386H, 386L,386N, 386R, 386S, 386T, 386V, 386W, 386Y, 387I, 387L, 388A, 388C, 388G,388H, 388L, 388P, 388S, 388T, 388W, 388Y, 389C, 389F, 389I, 389M, 389Q,389V, 390F, 390I, 390K, 390L, 390N, 390R, 390S, 390T, 390V, 390W, 390Y,391F, 391K, 391N, 391P, 391R, 391T, 391W, 391Y, 392A, 392C, 392D, 392E,392F, 392H, 392K, 392L, 392N, 392Q, 392R, 392S, 392V, 392Y, 393A, 393C,393D, 393F, 393G, 393H, 393I, 393L, 393Q, 393S, 393T, 393V, 393W, 393Y,394A, 394C, 394F, 394H, 394I, 394K, 394L, 394Q, 394V, 394W, 395F, 395G,395H, 395K, 395L, 395Q, 395R, 395S, 395T, 395V, 395W, 395Y, 396C, 396D,396S, 397C, 397D, 397F, 397G, 397H, 397I, 397L, 397P, 397S, 397T, 397V,397W, 398C, 398G, 398N, 398S, 398T, 398V, 399C, 399F, 399I, 399K, 399L,399R, 399S, 399T, 399V, 399W, 399Y, 400C, 400D, 400E, 400F, 400G, 400H,400I, 400K, 400L, 400M, 400Q, 400R, 400S, 400T, 400V, 400W, 400Y, 401A,401C, 401D, 401E, 401F, 401I, 401K, 401L, 401M, 401N, 401Q, 401R, 401S,401T, 401V, 401W, 401Y, 402A, 402C, 402D, 402E, 402F, 402G, 402H, 402I,402K, 402L, 402M, 402N, 402P, 402Q, 402R, 402T, 402V, 402W, 402Y, 403A,403C, 403H, 403I, 403M, 403V, 403W, 403Y, 404F, 404H, 404M, 404R, 404T,404V, 404W, 404Y, 405G, 405Q, 405S, 405T, 406L, 406T, 407F, 407G, 407H,407I, 407K, 407M, 407Q, 407R, 407S, 407T, 407V, 407W, 407Y, 408D, 408E,408F, 408N, 408V, 409C, 409F, 409I, 409L, 409R, 409T, 409V, 409W, 409Y,410V, 411E, 411F, 411M, 411Q, 411R, 411S, 411Y, 412N, 412T, 413C, 413F,413G, 413I, 413L, 413P, 413R, 413S, 413V, 413W, 413Y, 414H, 414L, 414N,414Q, 414T, 414V, 414W, 415D, 415E, 415G, 415I, 415R, 415V, 415W, 416F,416L, 416Q, 416Y, 417A, 417C, 417D, 417F, 417G, 417H, 417I, 417K, 417M,417N, 417Q, 418D, 418F, 418H, 418I, 418K, 418N, 418W, 418Y, 419E, 419F,419H, 419I, 419L, 419S, 419T, 420D, 420E, 420F, 420G, 420H, 420I, 420K,420L, 420Q, 420S, 420T, 420V, 420W, 420Y, 421C, 421L, 421M, 421S, 421T,422F, 422I, 422S, 422W, 423D, 423I, 423Q, 423R, 423T, 424M, 424Q, 424R,424V, 424Y, 425A, 425I, 425K, 425L, 425V, and 425Y.

In some embodiments, the substitution changes the amino acid residuepresent at position 153 to N, K or F, and the variant polypeptideexhibits increased ability to convert maltose and maltoheptaosesubstrates to glucose compared to the parental polypeptide. In someembodiments, the substitution changes the amino acid residue present atposition 153 to K, and the variant polypeptide exhibits increasedability to convert a DP7 substrate to glucose compared to the parentalpolypeptide.

In some embodiments, the substitution is selected from the groupconsisting of L142F, L142G, L142Q, L142S, L142W, L142Y, A214I, A214V,S245Y, Q126F, Q126L, Q126P, Q126V, S131L, and S254I, and wherein thesubstitution improves the starch liquefaction performance of the variantpolypeptides compared to the parental polypeptide of SEQ ID NO: 1. Insome embodiments, the substitution is selected from the group consistingof W60L, W60M, W60N, I100F, I100M, S105M, S105W, G207A, T270A, T270E,T270L, T270N, T270V, and T279A, and wherein the substitution improvesthe starch liquefaction performance of the variant polypeptides comparedto the parental polypeptide of SEQ ID NO: 2.

In some embodiments, the substitution is selected from the groupconsisting of 052D, 052E, 052I, 052K, 052L, 052N, 052Q, 052R, 052V,056D, 056E, 056I, 056K, 056L, 056N, 056Q, 056R, 056V, 089D, 089E, 089I,089K, 089L, 089N, 089Q, 089R, 089V, 152D, 152E, 152I, 152K, 152L, 152N,152Q, 152R, 152V, 153D, 153E, 153I, 153K, 153L, 153N, 153Q, 153R, 153V,201D, 201E, 201I, 201K, 201L, 201N, 201Q, 201R, 201V, 251D, 251E, 251I,251K, 251L, 251N, 251Q, 251R, 251V, 284D, 284E, 284I, 284K, 284L, 284N,284Q, 284R, 284V, 297D, 297E, 297I, 297K, 297L, 297N, 297Q, 297R, 297V,308D, 308E, 308I, 308K, 308L, 308N, 308Q, 308R, 308V, 321D, 321E, 321I,321K, 321L, 321N, 321Q, 321R, 321V, 328D, 328E, 328I, 328K, 328L, 328N,328Q, 328R, 328V, 347D, 347E, 347I, 347K, 347L, 347N, 347Q, 347R, 347V,357D, 357E, 357I, 357K, 357L, 357N, 357Q, 357R, 357V, 359D, 359E, 359I,359K, 359L, 359N, 359Q, 359R, 359V, 369D, 369E, 369I, 369K, 369L, 369N,369Q, 369R, 369V, 385D, 385E, 385I, 385K, 385L, 385N, 385Q, 385R, 385V,388D, 388E, 388I, 388K, 388L, 388N, 388Q, 388R, 388V, 391D, 391E, 391I,391K, 391L, 391N, 391Q, 391R, 391V, 400D, 400E, 400I, 400K, 400L, 400N,400Q, 400R, 400V, 416D, 416E, 416I, 416K, 416L, 416N, 416Q, 416R, and416V, which mutations have PI values >0.5 for both protein and activity.

In some embodiments, the variant polypeptide does not include amodification of the amino acid residue at a position selected from thegroup consisting of 75, 97, 101, 102, 120, 123, 133, 137, 182, 266, and306, of the parental polypeptide. In some embodiments, the variantpolypeptide does not include a modification of the amino acid residue ata position selected from the group consisting of 75 and 123, which weredetermined to be fully restrictive for performance in the truncatedparental polypeptide. In some embodiments, the variant polypeptide doesnot include a modification of the amino acid residue at a positionselected from the group consisting of 75, 97, 101, 102, 120, 133, 137,182, 266, and 306, which were determined to be fully restrictive forperformance in the full-length parental polypeptide.

In some embodiments, the parental polypeptide has at least 80% aminoacid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2.In some embodiments, the parental polypeptide has at least 90% aminoacid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2.In some embodiments, the parental polypeptide has at least 90% aminoacid sequence identity to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the characteristic that improves enzyme performanceis selected from the group consisting of increased thermal stability,increased specific activity, and increased protein expression.

In some embodiments, the characteristic that improves enzyme performanceis selected from the group consisting of increased thermal stability,increased specific activity, and increased protein expression.

Also provided is a starch processing composition comprising an AmyEpolypeptide (including a variant) and optionally a glucoamylase, apullulanase, a β-amylase, a fungal α-amylase, a protease, a cellulase, ahemicellulase, a lipase, a cutinase, an isoamylase, or a combinationthereof. In particular embodiments, the composition includes anadditional polypeptide having glucoamylase activity.

Also provided is a baking composition comprising an AmyE polypeptide(including a variant) in a solution or in a gel. A method of bakingcomprises adding the baking composition to a substance to be baked, andbaking the substance.

In another aspect, a cleaning composition is provided, comprising anAmyE polypeptide (including a variant) in an aqueous solution, andoptionally another enzyme, a detergent and/or a bleach agent. Thecleaning solution may used for laundering clothes, washing dishes, orcleaning other surfaces. In a related method, dishes, laundry, or othersurfaces are contacted with the cleaning composition for a timesufficient for the article to be cleaned.

In another aspect, a textile desizing composition is provided,comprising an AmyE polypeptide (including a variant) in an aqueoussolution, and optionally another enzyme. In a related method, a textileis contacted with the desizing composition for a time sufficient todesize the textile.

In another aspect, nucleic acids encoding AmyE variants, expressionvectors comprising such polynucleotides, and host cells that expressAmyE variants are provided. In yet another aspect, a nucleic acidcomplementary to a nucleic acid encoding any of the AmyE variants setforth herein is provided. Additionally, a nucleic acid capable ofhybridizing to a nucleic acid encoding any of the AmyE variants setforth herein, or the complement, thereof, is provided. In anotheraspect, compositions and methods involving a synthetic nucleic acidencoding any of the AmyE variants set forth herein, wherein the codonsare optionally optimized for expression in a particular host organism,are provided.

These and other aspects and embodiments of the compositions and methodswill be apparent from the following description and accompanyingFigures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a sequence alignment between AmyE having the amino acidsequence of SEQ ID NO: 1 (herein referred to as “AmyE full-length”) andAmyE having the amino acid sequence of SEQ ID NO: 3 (herein referred toas “Amy31A”). Differences in the amino acid sequences are shown in bold.Residues are numbered from the first amino acid in the mature form ofthe enzymes.

FIG. 2 depicts plasmid pME630-7, which comprises a polynucleotide(labeled “SAMY 425aa”) that encodes AmyE-tr (SEQ ID NO: 2). The plasmidcomprises a polynucleotide in-frame with the SAMY gene that encodes asignal sequence from B. licheniformis α-amylase (labeled “pre LAT”).

FIG. 3 depicts the relative specific activity of AmyE (SEQ ID NO: 1;“AmyE full length”), AmyE-tr (SEQ ID NO: 2; “AmyE truncated”), andAmy31A (SEQ ID NO: 3) toward an insoluble cornstarch substrate labeledwith Remazol Brilliant Blue (RBB). Hydrolysis of the substrate wascatalyzed for 30 minutes at 50° C. at pH 4.5, 5.0, or 5.6. Enzymeactivity was determined by the absorbance at 595 nm of released RBBlabel.

FIG. 4A depicts the viscosity (μNm) of 15 g ds starch substrate measuredas a function of time (minutes) in the presence of 0.7 mg AmyE-tr (SEQID NO: 2) at pH of 4.5 or 5.8. FIG. 4B depicts substrate viscosity as afunction of time in the presence of 2.2 mg AmyE (SEQ ID NO: 1) at pH of4.5 or 5.8. FIG. 4C depicts substrate viscosity as a function of time inthe presence of 1.4 mg Amy31A (SEQ ID NO: 3) at pH of 4.5 or 5.8. FIG.4D depicts substrate viscosity as a function of time in the presence of4.1 mg Amy31A at pH of 4.5 or 5.8.

FIG. 5A depicts differential scanning calorimetric (DSC) analysis ofexcess heat capacity functions of AmyE (SEQ ID NO: 1; “AmyE fulllength”) and AmyE-tr (SEQ ID NO: 2; AmyE truncated”) in the presence orabsence of 2 mM Ca²⁺. FIG. 5B depicts DSC analysis of Amy31A (SEQ ID NO:3) in the presence or absence of 2 mM Ca²⁺. FIG. 5C depicts DSC analysisof Geobacillus stearothermophilus α-amylase (AmyS) in the presence orabsence of 2 mM Ca²⁺.

FIG. 6 depicts the results of glucose production from a maltosesubstrate by AmyE-tr (SEQ ID NO: 2) compared to AmyE position Q153variants and truncated AmyS (SEQ ID NO: 4).

FIG. 7 depicts the results of glucose production from a maltoheptaosesubstrate by AmyE-tr (SEQ ID NO: 2) compared to AmyE position Q153variants and truncated AmyS (SEQ ID NO: 4).

FIG. 8 depicts breakdown products detected by HPLC following a 0 h (toppanel) and 72 h incubation (bottom panel) of AmyE-tr with maltoheptaose(DP7).

FIG. 9 depicts breakdown products detected by HPLC following a 0 h, 2 h,4 h, and 24 h (panels from top to bottom) incubation of AmyS with a DP7substrate.

FIG. 10 depicts products formed by incubating AmyE-tr Q153K variant withmaltoheptaose (DP7). HPLC traces shown from top to bottom correspond totime 0 h, 1 h, 2 h, 3 h, and 24 h.

FIG. 11 shows ethanol formation by truncated AmyE and SPEZYME® Xtraamylase in conventional fermentation at pH 4.3 and 5.8.

FIG. 12 shows hydrolysis of insoluble granular (uncooked) starch intoethanol by full length (FL) and truncated (tr) AmyE compared to AkAAalone or Stargen™ 002 at pH 4.3 and pH 5.8.

FIG. 13 shows glucose formation by B. subtilis AmyE full-length, AmyEtruncated, and Amy 31A in comparison to AmyS at pH 4.5 and 5.6.

FIG. 14 shows the results of incubation of full length AmyE with rawstarch (corn flour) and detection of oligosaccharides produced over time(0, 30, 90 minutes) by HPLC detection.

FIG. 15 shows the peak and final viscosity values obtained in a starchhydrolysis assay using AmyE polypeptides.

FIG. 16 shows the results of a bread staling assay using an AmyEpolypeptide.

BRIEF DESCRIPTION OF THE SEQUENCES

The following sequences are referred to in the application:

SEQ ID NO: 1: Full length Bacillus subtilis AmyE amino acid sequence. The nativesignal sequence is not shown. 1LTAPSIKSGT ILHAWNWSFN TLKHNMKDIH DAGYTAIQTS PINQVKEGNQ 51GDKSMSNWYW LYQPTSYQIG NRYLGTEQEF KEMCAAAEEY GIKVIVDAVI 101NHTTSDYAAI SNEVKSIPNW THGNTQIKNW SDRWDVTQNS LLGLYDWNTQ 151NTQVQSYLKR FLDRALNDGA DGFRFDAAKH IELPDDGSYG SQFWPNITNT 201SAEFQYGEIL QDSASRDAAY ANYMDVTASN YGHSIRSALK NRNLGVSNIS 251HYASDVSADK LVTWVESHDT YANDDEESTW MSDDDIRLGW AVIASRSGST 301PLFFSRPEGG GNGVRFPGKS QIGDRGSALF EDQAITAVNR FHNVMAGQPE 351ELSNPNGNNQ IFMNQRGSHG VVLANAGSSS VSINTATKLP DGRYDNKAGA 401GSFQVNDGKL TGTINARSVA VLYPDDIAKA PHVFLENYKT GVTHSFNDQL 451TITLRADANT TKAVYQINNG PETAFKDGDQ FTIGKGDPFG KTYTIMLKGT 501NSDGVTRTEK YSFVKRDPAS AKTIGYQNPN HWSQVNAYIY KHDGSRVIEL 551TGSWPGKPMT KNADGIYTLT LPADTDTTNA KVIFNNGSAQ VPGQNQPGFD 601YVLNGLYNDS GLSGSLPHSEQ ID NO: 2: Truncated Bacillus subtilis AmyE (AmyE-tr) amino acid sequence.The native signal sequence is not shown. 1LTAPSIKSGT ILHAWNWSFN TLKHNMKDIH DAGYTAIQTS PINQVKEGNQ 51GDKSMSNWYW LYQPTSYQIG NRYLGTEQEF KEMCAAAEEY GIKVIVDAVI 101NHTTSDYAAI SNEVKSIPNW THGNTQIKNW SDRWDVTQNS LLGLYDWNTQ 151NTQVQSYLKR FLDRALNDGA DGFRFDAAKH IELPDDGSYG SQFWPNITNT 201SAEFQYGEIL QDSASRDAAY ANYMDVTASN YGHSIRSALK NRNLGVSNIS 251HYASDVSADK LVTWVESHDT YANDDEESTW MSDDDIRLGW AVIASRSGST 301PLFFSRPEGG GNGVRFPGKS QIGDRGSALF EDQAITAVNR FHNVMAGQPE 351ELSNPNGNNQ IFMNQRGSHG VVLANAGSSS VSINTATKLP DGRYDNKAGA 401GSFQVNDGKL TGTINARSVA VLYPDSEQ ID NO: 3: Bacillus subtilis α-amylase variant Amy31A amino acid sequence(UniProtKB/TrEMBL Accession No. O82953). The native signal sequence is shown in bold.1 MFEKRFKTSL LPLFAGFLLL FHLVLSGPAA ANAETANKSN KVTASSVKNG 51TILHAWNWSF NTLTQNMKDI RDAGYAAIQT SPINQVKEGN QGDKSMSNWY 101WLYQPTSYQI GNRYLGTEQE FKDMCAAAEK YGVKVIVDAV VNHTTSDYGA 151ISDEIKRIPN WTHGNTQIKN WSDRWDITQN ALLGLYDWNT QNTEVQAYLK 201GFLERALNDG ADGFRYDAAK HIELPDDGNY GSQFWPNITN TSAEFQYGEI 251LQDSASRDTA YANYMNVTAS NYGHSIRSAL KNRILSVSNI SHYASDVSAD 301KLVTWVESHD TYANDDEEST WMSDDDIRLG WAVIGSRSGS TPLFFSRPEG 351GGNGVRFPGK SQIGDRGSAL FKDQAITAVN QFHNEMAGQP EELSNPNGNN 401QIFMNQRGSK GVVLANAGSS SVTINTSTKL PDGRYDNRAG AGSFQVANGK 451LTGTINARSA AVLYPDDIGN APHVFLENYQ TEAVHSFNDQ LTVTLRANAK 501TTKAVYQINN GQETAFKDGD RLTIGKEDPI GTTYNVKLTG TNGEGASRTQ 551EYTFVKKDPS QTNIIGYQNP DHWGNVNAYI YKHDGGGAIE LTGSWPGKAM 601TKNADGIYTL TLPANADTAD AKVIFNNGSA QVPGQNHPGF DYVQNGLYNN 651 SGLNGYLPHSEQ ID NO: 4: Truncated Geobacillus stearothermophilus α-amylase (AmyS) proteinsequence (SPEZYME ® Xtra amylase). The signal sequence is shown in bold.1 MLTFHRIIRK GWMFLLAFLL TASLFCPTGQ HAKAAAPFNG TMMQYFEWYL 51PDDGTLWTKV ANEANNLSSL GITALWLPPA YKGTSRSDVG YGVYDLYDLG 101EFNQKGTVRT KYGTKAQYLQ AIQAAHAAGM QVYADVVFDH KGGADGTEWV 151DAVEVNPSDR NQEISGTYQI QAWTKFDFPG RGNTYSSFKW RWYHFDGVDW 201DESRKLSRIY KFIGKAWDWE VDTENGNYDY LMYADLDMDH PEVVTELKNW 251GKWYVNTTNI DGFRLDAVKH IKFSFFPDWL SYVRSQTGKP LFTVGEYWSY 301DINKLHNYIT KTNGTMSLFD APLHNKFYTA SKSGGAFDMR TLMTNTLMKD 351QPTLAVTFVD NHDTEPGQAL QSWVDPWFKP LAYAFILTRQ EGYPCVFYGD 401YYGIPQYNIP SLKSKIDPLL IARRDYAYGT QHDYLDHSDI IGWTREGVTE 451KPGSGLAALI TDGPGGSKWM YVGKQHAGKV FYDLTGNRSD TVTINSDGWG 501EFKVNGGSVS VWVPRKTTSEQ ID NO: 5: Nucleotide sequence encoding the AmyE of SEQ ID NO: 1.CTTACAGCACCGTCGATCAAAAGCGGAACCATTCTTCATGCATGGAATTGGTCGTTCAATACGTTAAAACACAATATGAAGGATATTCATGATGCAGGATATACAGCCATTCAGACATCTCCGATTAACCAAGTAAAGGAAGGGAATCAAGGAGATAAAAGCATGTCGAACTGGTACTGGCTGTATCAGCCGACATCGTATCAAATTGGCAACCGTTACTTAGGTACTGAACAAGAATTTAAAGAAATGTGTGCAGCCGCTGAAGAATATGGCATAAAGGTCATTGTTGACGCGGTCATCAATCATACCACCAGTGATTATGCCGCGATTTCCAATGAGGTTAAGAGTATTCCAAACTGGACACATGGAAACACACAAATTAAAAACTGGTCTGATCGATGGGATGTCACGCAGAATTCATTGCTCGGGCTGTATGACTGGAATACACAAAATACACAAGTACAGTCCTATCTGAAACGGTTCTTAGACAGGGCATTGAATGACGGGGCAGACGGTTTTCGATTTGATGCCGCCAAACATATAGAGCTTCCAGATGATGGCAGTTACGGCAGTCAATTTTGGCCGAATATCACAAATACATCAGCAGAGTTCCAATACGGAGAAATCCTTCAGGATAGTGCCTCCAGAGATGCTGCATATGCGAATTATATGGATGTGACAGCGTCTAACTATGGGCATTCCATAAGGTCCGCTTTAAAGAATCGTAATCTGGGCGTGTCGAATATCTCCCACTATGCATCTGATGTGTCTGCGGACAAGCTAGTGACATGGGTAGAGTCGCATGATACGTATGCCAATGATGATGAAGAGTCGACATGGATGAGCGATGATGATATCCGTTTAGGCTGGGCGGTGATAGCTTCTCGTTCAGGCAGTACGCCTCTTTTCTTTTCCAGACCTGAGGGAGGCGGAAATGGTGTGAGGTTCCCGGGGAAAAGCCAAATAGGCGATCGCGGGAGTGCTTTATTTGAAGATCAGGCTATCACTGCGGTCAATAGATTTCACAATGTGATGGCTGGACAGCCTGAGGAACTCTCGAACCCGAATGGAAACAACCAGATATTTATGAATCAGCGCGGCTCACATGGCGTTGTGCTGGCAAATGCAGGTTCATCCTCTGTCTCTATCAATACGGCAACAAAATTGCCTGATGGCAGGTATGACAATAAAGCTGGAGCGGGTTCATTTCAAGTGAACGATGGTAAACTGACAGGCACGATCAATGCCAGGTCTGTAGCTGTGCTTTATCCTGATGATATTGCAAAAGCGCCTCATGTTTTCCTTGAGAATTACAAAACAGGTGTAACACATTCTTTCAATGATCAACTGACGATTACCTTGCGTGCAGATGCGAATACAACAAAAGCCGTTTATCAAATCAATAATGGACCAGAGACGGCGTTTAAGGATGGAGATCAATTCACAATCGGAAAAGGAGATCCATTTGGCAAAACATACACCATCATGTTAAAAGGAACGAACAGTGATGGTGTAACGAGGACCGAGAAATACAGTTTTGTTAAAAGAGATCCAGCGTCGGCCAAAACCATCGGCTATCAAAATCCGAATCATTGGAGCCAGGTAAATGCTTATATCTATAAACATGATGGGAGCCGAGTAATTGAATTGACCGGATCTTGGCCTGGAAAACCAATGACTAAAAATGCAGACGGAATTTACACGCTGACGCTGCCTGCGGACACGGATACAACCAACGCAAAAGTGATTTTTAATAATGGCAGCGCCCAAGTGCCCGGTCAGAATCAGCCTGGCTTTGATTACGTGCTAAATGGTTTATATAATGACTCGGGCTTAAGCGGTTCTCTTCCCCATSEQ ID NO: 6: Nucleotide sequence encoding AmyE-tr (SEQ ID NO: 2).CTTACAGCACCGTCGATCAAAAGCGGAACCATTCTTCATGCATGGAATTGGTCGTTCAATACGTTAAAACACAATATGAAGGATATTCATGATGCAGGATATACAGCCATTCAGACATCTCCGATTAACCAAGTAAAGGAAGGGAATCAAGGAGATAAAAGCATGTCGAACTGGTACTGGCTGTATCAGCCGACATCGTATCAAATTGGCAACCGTTACTTAGGTACTGAACAAGAATTTAAAGAAATGTGTGCAGCCGCTGAAGAATATGGCATAAAGGTCATTGTTGACGCGGTCATCAATCATACCACCAGTGATTATGCCGCGATTTCCAATGAGGTTAAGAGTATTCCAAACTGGACACATGGAAACACACAAATTAAAAACTGGTCTGATCGATGGGATGTCACGCAGAATTCATTGCTCGGGCTGTATGACTGGAATACACAAAATACACAAGTACAGTCCTATCTGAAACGGTTCTTAGACAGGGCATTGAATGACGGGGCAGACGGTTTTCGATTTGATGCCGCCAAACATATAGAGCTTCCAGATGATGGCAGTTACGGCAGTCAATTTTGGCCGAATATCACAAATACATCAGCAGAGTTCCAATACGGAGAAATCCTTCAGGATAGTGCCTCCAGAGATGCTGCATATGCGAATTATATGGATGTGACAGCGTCTAACTATGGGCATTCCATAAGGTCCGCTTTAAAGAATCGTAATCTGGGCGTGTCGAATATCTCCCACTATGCATCTGATGTGTCTGCGGACAAGCTAGTGACATGGGTAGAGTCGCATGATACGTATGCCAATGATGATGAAGAGTCGACATGGATGAGCGATGATGATATCCGTTTAGGCTGGGCGGTGATAGCTTCTCGTTCAGGCAGTACGCCTCTTTTCTTTTCCAGACCTGAGGGAGGCGGAAATGGTGTGAGGTTCCCGGGGAAAAGCCAAATAGGCGATCGCGGGAGTGCTTTATTTGAAGATCAGGCTATCACTGCGGTCAATAGATTTCACAATGTGATGGCTGGACAGCCTGAGGAACTCTCGAACCCGAATGGAAACAACCAGATATTTATGAATCAGCGCGGCTCACATGGCGTTGTGCTGGCAAATGCAGGTTCATCCTCTGTCTCTATCAATACGGCAACAAAATTGCCTGATGGCAGGTATGACAATAAAGCTGGAGCGGGTTCATTTCAAGTGAACGATGGTAAACTGACAGGCACGATCAATGCCAGGTCTGTAGCTGTGCTTTATCCTGATSEQ ID NO: 7: Nucleotide sequence encoding B. subtilis Amy31A (SEQ ID NO: 3).TCTGTTAAAAACGGCACTATTCTGCATGCATGGAACTGGAGCTTTAACACGCTGACCCAGAACATGAAAGATATTCGTGACGCGGGCTATGCTGCGATCCAAACCAGCCCTATCAACCAGGTCAAAGAAGGCAACCAAGGCGACAAATCCATGTCCAACTGGTACTGGCTGTATCAACCGACGTCCTATCAGATTGGCAACCGTTATCTGGGCACGGAGCAAGAGTTCAAAGACATGTGTGCTGCGGCTGAGAAATATGGTGTGAAAGTTATCGTGGACGCTGTGGTAAACCACACGACCTCTGATTATGGTGCTATTAGCGACGAGATTAAACGTATTCCAAATTGGACCCATGGTAATACCCAGATCAAAAATTGGAGCGACCGCTGGGACATTACCCAGAATGCGCTGCTGGGTCTGTATGACTGGAACACGCAAAACACCGAAGTACAGGCATATCTGAAGGGCTTCCTGGAACGCGCTCTGAACGATGGTGCTGATGGTTTTCGCTACGACGCCGCAAAGCATATTGAGCTGCCGGATGACGGCAACTACGGTTCCCAATTCTGGCCGAACATCACCAACACCTCTGCCGAATTCCAGTACGGCGAGATCCTGCAAGACTCCGCGAGCCGTGACACCGCTTATGCCAACTATATGAACGTAACTGCCTCTAACTATGGCCATTCCATTCGTTCTGCGCTGAAAAATCGTATCCTGTCCGTGTCCAATATCTCCCACTATGCATCCGACGTTTCTGCTGACAAACTGGTAACTTGGGTCGAGTCTCACGACACCTATGCAAATGATGACGAGGAGAGCACCTGGATGAGCGATGATGATATTCGTCTGGGTTGGGCGGTTATTGGTTCTCGCTCTGGTTCTACTCCGCTGTTCTTTAGCCGTCCGGAAGGTGGCGGCAATGGCGTTCGTTTCCCGGGTAAATCTCAAATTGGTGATCGTGGCTCTGCACTGTTTAAAGATCAAGCTATTACGGCGGTGAATCAGTTCCATAATGAGATGGCAGGTCAACCTGAAGAACTGTCCAATCCAAACGGTAACAACCAAATCTTCATGAACCAGCGTGGCAGCAAAGGCGTCGTCCTGGCGAACGCCGGTAGCTCTTCTGTTACCATCAACACGTCTACCAAACTGCCAGACGGCCGCTATGATAACCGTGCGGGTGCTGGTTCCTTTCAGGTAGCCAACGGCAAGCTGACGGGCACCATCAACGCTCGTTCTGCTGCTGTTCTGTACCCGGACGACATTGGCAACGCTCCGCACGTGTTCCTGGAGAATTACCAGACCGAAGCGGTACATAGCTTTAATGACCAGCTGACCGTCACTCTGCGTGCCAACGCAAAAACCACGAAAGCAGTCTATCAGATCAATAATGGTCAAGAAACTGCTTTCAAGGATGGCGACCGTCTGACTATTGGTAAGGAGGACCCGATTGGCACCACTTATAACGTTAAACTGACTGGCACCAATGGCGAGGGCGCTAGCCGCACTCAAGAGTATACGTTCGTAAAGAAAGACCCGTCTCAAACCAACATCATCGGTTACCAGAATCCTGACCACTGGGGTAATGTGAACGCTTACATCTATAAACATGATGGTGGCGGTGCTATCGAACTGACCGGCTCTTGGCCAGGTAAAGCCATGACGAAAAACGCGGATGGCATCTATACCCTGACCCTGCCGGCCAATGCGGATACCGCAGATGCGAAGGTTATCTTCAATAACGGCTCCGCGCAGGTTCCGGGCCAAAACCATCCGGGCTTTGACTACGTACAAAATGGTCTGTATAACAACTCTGGCCTGAACGGTTACCTGCCGCACSEQ ID NO: 8: Nucleotide sequence encoding Geobacillus stearothermophilus AmyS(SEQ ID NO: 4).GCCGCACCGTTTAACGGTACCATGATGCAGTATTTTGAATGGTACTTGCCGGATGATGGCACGTTATGGACCAAAGTGGCCAATGAAGCCAACAACTTATCCAGCCTTGGCATCACCGCTCTTTGGCTGCCGCCCGCTTACAAAGGAACAAGCCGCAGCGACGTAGGGTACGGAGTATACGACTTGTATGACCTCGGCGAATTCAATCAAAAAGGGACCGTCCGCACAAAATATGGAACAAAAGCTCAATATCTTCAAGCCATTCAAGCCGCCCACGCCGCTGGAATGCAAGTGTACGCCGATGTCGTGTTCGACCATAAAGGCGGCGCTGACGGCACGGAATGGGTGGACGCCGTCGAAGTCAATCCGTCCGACCGCAACCAAGAAATCTCGGGCACCTATCAAATCCAAGCATGGACGAAATTTGATTTTCCCGGGCGGGGCAACACCTACTCCAGCTTTAAGTGGCGCTGGTACCATTTTGACGGCGTTGACTGGGACGAAAGCCGAAAATTAAGCCGCATTTACAAATTCATCGGCAAAGCGTGGGATTGGGAAGTAGACACAGAAAACGGAAACTATGACTACTTAATGTATGCCGACCTTGATATGGATCATCCCGAAGTCGTGACCGAGCTGAAAAACTGGGGGAAATGGTATGTCAACACAACGAACATTGATGGGTTCCGGCTTGATGCCGTCAAGCATATTAAGTTCAGTTTTTTTCCTGATTGGTTGTCGTATGTGCGTTCTCAGACTGGCAAGCCGCTATTTACCGTCGGGGAATATTGGAGCTATGACATCAACAAGTTGCACAATTACATTACGAAAACAAACGGAACGATGTCTTTGTTTGATGCCCCGTTACACAACAAATTTTATACCGCTTCCAAATCAGGGGGCGCATTTGATATGCGCACGTTAATGACCAATACTCTCATGAAAGATCAACCGACATTGGCCGTCACCTTCGTTGATAATCATGACACCGAACCCGGCCAAGCGCTGCAGTCATGGGTCGACCCATGGTTCAAACCGTTGGCTTACGCCTTTATTCTAACTCGGCAGGAAGGATACCCGTGCGTCTTTTATGGTGACTATTATGGCATTCCACAATATAACATTCCTTCGCTGAAAAGCAAAATCGATCCGCTCCTCATCGCGCGCAGGGATTATGCTTACGGAACGCAACATGATTATCTTGATCACTCCGACATCATCGGGTGGACAAGGGAAGGGGTCACTGAAAAACCAGGATCCGGGCTGGCCGCACTGATCACCGATGGGCCGGGAGGAAGCAAATGGATGTACGTTGGCAAACAACACGCTGGAAAAGTGTTCTATGACCTTACCGGCAACCGGAGTGACACCGTCACCATCAACAGTGATGGATGGGGGGAATTCAAAGTCAATGGCGGTTCGGTTTCGGTTTGGGTTCCTAGAAAAACGACCSEQ ID NO: 9: Native signal sequence of the AmyE of SEQ ID NO: 1.MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASAETANKSNESEQ ID NO: 10: Primer PSTAMYE-F 5′CTTCTTGCTGCCTCATTCTGCAGCTTCAGCACTTACAGCACCGTCGATCAAAAGCGG AACSEQ ID NO: 11: Primer AMYENOPST-R 5′CTGGAGGCACTATCCTGAAGGATTTCTCCGTATTGGAACTCTGCTGATGTATTTGTGSEQ ID NO: 12: Primer AMYENOPST-F 5′CACAAATACATCAGCAGAGTTCCAATACGGAGAAATCCTTCAGGATAGTGCCTCCAGSEQ ID NO: 13: Primer HPAIAMYE-R 5′CAGGAAATCCGTCCTCTGTTAACTCAATGGGGAAGAGAACCGCTTAAGCCCGAGTCSEQ ID NO: 14: Primer HPAIAMYE466-R 5′CAGGAAATCCGTCCTCTGTTAACTCAATCAGGATAAAGCACAGCTACAGACCTGGSEQ ID NO: 15: Primer AMYE SEQ-F1 5′ TACACAAGTACAGTCCTATCTGSEQ ID NO: 16: Primer AMYE SEQ-F2 5′ CATCCTCTGTCTCTATCAATACSEQ ID NO: 17: BP-17 variant of Buttiauxiella phytaseNDTPASGYQV EKVVILSRHG VRAPTKMTQT MRDVTPNTWP EWPVKLGYITPRGEHLISLM GGFYRQKFQQ QGILSQGSCP TPNSIYVWAD VDQRTLKTGEAFLAGLAPQC GLTIHHQQNL EKADPLFHPV KAGTCSMDKT QVQQAVEKEAQTPIDNLNQH YIPFLALMNT TLNFSTSAWC QKHSADKSCD LGLSMPSKLSIKDNGNKVAL DGAIGLSSTL AEIFLLEYAQ GMPQAAWGNI HSEQEWASLLKLHNVQFDLM ARTPYIARHN GTPLLQAISN ALNPNATESK LPDISPDNKILFIAGHDTNI ANIAGMLNMR WTLPGQPDNT PPGGALVFER LADKSGKQYVSVSMVYQTLE QLRSQTPLSL NQPAGSVQLK IPGCNDQTAE GYCPLSTFTR VVSQSVEPGC QLQ

DETAILED DESCRIPTION

The present compositions and methods relate to an α-amylase fromBacillus subtilis (AmyE) and variants thereof (collectively referred toas AmyE polypeptides), which offer certain advantages compared to otherα-amylases. For example, AmyE polypeptides exhibit high specificactivity for starch substrates at an acidic pH, allowing AmyEpolypeptides to be used for starch liquefaction under conditions thatare also suitable for saccharification. This eliminates the need toadjust the pH of a starch slurry between liquefaction andsaccharification. AmyE polypeptides also possesses glucoamylaseactivity, eliminating or reducing the need for the use of a separateglucoamylase to perform saccharifaction. In addition, AmyE polypeptidesrequire little or no calcium for thermal stability, eliminating the needto subsequently remove added calcium prior to performing isomerization,thereby eliminating at least one step required for the production ofhigh fructose corn syrup.

These and other features of the compositions and methods are describedin more detail, below.

1. Definitions and Abbreviations

Unless defined otherwise, all technical and scientific terms andabbreviations should be accorded their ordinary meanings as understoodby one of ordinary skill in the art. The following terms andabbreviations are defined for clarity.

1.1. Definitions

As used herein the term “starch” refers to any material comprised of thecomplex polysaccharide carbohydrates of plants, comprised of amylose andamylopectin with the formula (C₆H₁₀O₅)_(x), wherein X can be any number.In particular, the term refers to any plant-based material including butnot limited to grains, grasses, tubers and roots and more specificallywheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, potato,sweet potato, and tapioca.

As used herein the term “oligosaccharide” refers to a carbohydratemolecule composed of 3-20 monosaccharides.

As used herein, an “amylase” refers to an enzyme capable of catalyzingthe degradation of starch. Generally, α-amylases (EC 3.2.1.1;α-D-(1→4)-glucan glucanohydrolase) are endo-acting enzymes that cleaveα-D-(1→4)O-glycosidic linkages within the starch molecule in a randomfashion. In contrast, the exo-acting amylolytic enzymes, such asβ-amylases (EC 3.2.1.2; α-D-(1→4)-glucan maltohydrolase), and someproduct-specific amylases like maltogenic α-amylase (EC 3.2.1.133),cleave the starch molecule from the non-reducing end of the substrate.β-amylases, α-glucosidases (EC 3.2.1.20; α-D-glucoside glucohydrolase),glucoamylases (EC 3.2.1.3; α-D-(1→4)-glucan glucohydrolase), andproduct-specific amylases can produce malto-oligosaccharides of aspecific length from starch. As used herein, amylases include any/allamylases, including glucoamylases, α-amylases, β-amylases and wild-typeα-amylases, such as those of Bacillus sp., e.g., B. licheniformis and B.subtilis, while α-amylases include the aforementioned subset of theseenzymes.

As used herein, “α-amylase variants,” and similar phrases, refer tovariants/mutants of a reference α-amylase, which includes an amino acidsubstitution, insertion, and/or deletion with respect to the parent(wild-type; reference) amino acid sequence of the reference α-amylase.The term “variant” is used interchangeably with the term “mutant.” Thevariant α-amylase may include mutations in the signal sequence withrespect to parent signal sequence. In addition, the variant α-amylasecan be in the form of a fusion protein containing a heterologousα-amylase signal sequence, such as from B. licheniformis (LAT).

A “parent nucleic acid/polynucleotide,” “wild-type nucleicacid/polynucleotide,” or “reference nucleic acid/polynucleotide,” refersto a nucleic acid sequence encoding a parent polypeptide, and a nucleicacid complementary thereto.

A “variant nucleic acid/polynucleotide” refers to a nucleic acidsequence encoding a variant polypeptide or a nucleic acid complementarythereto, or a polynucleotide sequence having at least one basesubstitution, insertion, or deletion with respect to a parentpolynucleotide sequence or a nucleic acid complementary thereto. Wherespecified such nucleic acids may include those having a specified degreeof homology to a reference sequence, or that are capable of hybridizingto a reference sequence, for example, under stringent conditions [e.g.,50° C. and 0.2×SSC (1×SSC=0.15 M NaCl, 0.015 M Na₃ citrate, pH 7.0)] orhighly stringent conditions [e.g., 65° C. and 0.1×SSC (1×SSC=0.15 MNaCl, 0.015 M Na₃ citrate, pH 7.0)]. A variant nucleic acid may beoptimized to reflect preferred codon usage for a specified hostorganisms, such as the methylotrophic yeasts (e.g., Pichia, Hansenula,etc) or filamentous fungi (e.g., Trichoderma (e.g., T. reesei), etc) orother expression hosts (e.g., Bacillus, Streptomyces, etc.).

A “signal sequence” is a sequence of amino acids attached to theN-terminal portion of a protein, which facilitates the secretion of theprotein outside the cell. The “signal sequence,” may also be referred toas a “leader sequence” or a “pro-sequence.”

As used herein, the “immature” or “full-length (FL)” form of an amylaseincludes the signal peptide. The immature form may include otherpost-translational modifications.

As used herein, the “mature” form of an extracellular protein (such asan amylase) lacks the signal sequence. The signal sequence may becleaved off during the secretion process. Alternatively, a polypeptidemay be expressed in its mature form, e.g., as an intracellular protein,or synthesized in its mature form.

As used herein, a “truncated” form of AmyE (i.e., “AmyE-tr”) refers toan AmyE polypeptide with a deletion of all or part of the C-terminalstarch binding domain. In the AmyE-tr of SEQ ID NO: 2, for example, theAmyE of SEQ ID NO: 1 is truncated at residue D425. A 2.5 Å resolutioncrystal structure of this AmyE-tr is available at Protein DatabankAccession No. 1BAG, which is disclosed in Fujimoto et al., “Crystalstructure of a catalytic-site mutant alpha-amylase from B. subtiliscomplexed with maltopentaose,” J. Mol. Biol. 277: 393-407 (1998).AmyE-tr may be truncated at other positions, e.g., Y423, P424, D426 orI427 of the AmyE of SEQ ID NO: 1, provided all or part of the C-terminalstarch binding domain is removed.

The term “recombinant,” when used in reference to a subject cell,nucleic acid, protein or vector, indicates that the subject has beenmodified by the introduction of a heterologous nucleic acid or proteinor the alteration of a native nucleic acid or protein, or that the cellis derived from a cell so modified. Thus, for example, recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise abnormallyexpressed, under expressed or not expressed at all.

The terms “recovered,” “isolated,” and “separated,” refer to a compound,protein, cell, nucleic acid or amino acid that are removed from at leastone component with which it is naturally associated and found in nature.

As used herein, the term “purified” refers to material (e.g., anisolated polypeptide or polynucleotide) that is in a relatively purestate, e.g., at least about 90% pure, at least about 95% pure, at leastabout 98% pure, or even at least about 99% pure.

The terms “thermostable” and “thermostability” refer to the ability ofan enzyme to retain activity after exposure to an elevated temperature.The thermostability of an enzyme, such as an α-amylase enzymes, ismeasured by its half-life (t_(1/2)) given in minutes, hours, or days,during which half the enzyme activity is lost under defined conditions.The half-life may be calculated by measuring residual α-amylase activityfollowing exposure to (i.e., challenge by) an elevated temperature.

A “pH range” refers to the range of pH values under which an enzymeexhibits catalytic activity.

As used herein, the terms “pH stable” and “pH stability” relate to theability of an enzyme to retain activity over a wide range of pH valuesfor a predetermined period of time (e.g., 15 min., 30 min., 1 hour, andthe like).

As used herein, the term “amino acid sequence” is synonymous with theterms “polypeptide,” “protein,” and “peptide,” and are usedinterchangeably. Where such amino acid sequence exhibit activity, theymay be referred to as an “enzyme.” The conventional one-letter orthree-letter code for amino acid residues are used herein.

The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, andsynthetic molecules capable of encoding a polypeptide. Nucleic acids maybe single stranded or double stranded, and may be chemicalmodifications. The terms “nucleic acid” and “polynucleotide” are usedinterchangeably. Because the genetic code is degenerate, more than onecodon may be used to encode a particular amino acid, and the presentcompositions and methods encompass nucleotide sequences which encode aparticular amino acid sequence. Unless otherwise indicated, nucleicacids are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation,respectively.

The term “homologue” refers to an amino acid or nucleotide sequencehaving a certain degree of identity to a reference amino acid ornucleotide sequences, or another specified common structural orfunctional feature. A homologous sequence is taken to include an aminoacid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%identical to the subject sequence, using conventional sequence alignmenttools (e.g., Clustal, BLAST, and the like). Typically, homologues willinclude the same active site residues as the subject amino acidsequence, unless otherwise specified.

As used herein, “hybridization” refers to the process by which onestrand of nucleic acid base pairs with a complementary strand, as occursduring blot hybridization techniques and PCR techniques.

As used herein, a “degenerate sequence” in a nucleic acid contains isone in which a plurality of nucleotide sequences encode the same codon,i.e., due to the degeneracy of the genetic code. Degenerate sequencesmay be selected for optimal expression of an encoded polypeptide in aparticular host organism, e.g., as a consequence of codon preferences.

As used herein, a “synthetic” molecule is produced by in vitro chemicalor enzymatic synthesis rather than by an organism.

As used herein, the terms “transformed,” “stably transformed,” and“transgenic,” used with reference to a cell means that the cell has anon-native (e.g., heterologous) nucleic acid sequence integrated intoits genome or carried as an episomal plasmid that is maintained throughmultiple generations.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, “transformation” or“transduction,” as known in the art.

A “host strain” or “host cell” is an organism into which an expressionvector, phage, virus, or other DNA construct including a polynucleotideencoding a polypeptide of interest (e.g., a variant α-amylase) has beenintroduced. Exemplary host strains are bacterial cells. The term “hostcell” includes protoplasts created from cells, such as those of aBacillus sp.

The term “heterologous” with reference to a polynucleotide or proteinrefers to a polynucleotide or protein that does not naturally occur in ahost cell.

The term “endogenous” with reference to a polynucleotide or proteinrefers to a polynucleotide or protein that occurs naturally in the hostcell.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

A “selective marker” or “selectable marker” refers to a gene capable ofbeing expressed in a host to facilitate selection of host cells carryingthe gene. Examples of selectable markers include but are not limited toantimicrobials (e.g., hygromycin, bleomycin, or neomycin) and/or genesthat confer a metabolic advantage, such as a nutritional advantage onthe host cell.

“Culturing” refers to growing a population of microbial cells undersuitable conditions in a liquid or solid medium. Culturing includesfermentative bioconversion of a starch substrate containing granularstarch to an end-product (typically in a vessel or reactor).

“Fermentation” is the enzymatic breakdown of organic substances bymicroorganisms to produce simpler organic compounds. While fermentationgenerally occurs under anaerobic conditions it is not intended that theterm be solely limited to strict anaerobic conditions, as fermentationalso occurs in the presence of oxygen.

A “gene” refers to a DNA segment that is involved in producing apolypeptide, and includes coding regions, regions preceding andfollowing the coding regions, and, intervening sequences (introns)between individual coding segments (exons).

A “vector” refers to a polynucleotide sequence designed to introducenucleic acids into one or more cell types. Vectors include cloningvectors, expression vectors, shuttle vectors, plasmids, phage particles,cassettes and the like.

An “expression vector” refers to a DNA construct comprising a DNAsequence encoding a polypeptide of interest, which is operably linked toa suitable control sequence capable of effecting expression of the DNAin a suitable host. Such control sequences may include a promoter toeffect transcription, an optional operator sequence to controltranscription, a sequence encoding suitable ribosome binding sites onthe mRNA, enhancers and sequences which control termination oftranscription and translation.

A “promoter” is a regulatory sequence that is involved in binding RNApolymerase to initiate transcription of a gene. The promoter may be aninducible promoter or a constitutive promoter. An exemplary promoter isthe Bacillus licheniformis α-amylase (AmyL) promoter.

The term “operably linked” means that specified components are in arelationship (including but not limited to juxtaposition) permittingthem to function in an intended manner. For example, a regulatorysequence is operably linked to a coding sequence such that expression ofthe coding sequence is under control of the regulatory sequences.

The term, “under transcriptional control” means that transcription of apolynucleotide sequence, usually a DNA sequence, depends on its beingoperably linked to an element which contributes to the initiation of, orpromotes transcription.

The term “under translational control” means that translation of apolynucleotide sequence, usually an RNA sequence, into a polypeptidedepends on its being operably linked to an element which contributes tothe initiation of, or promotes translation.

As used herein, “biologically active” refer to a sequence having aspecified biological activity, such an enzymatic activity. In the caseof the present amylases, the activity is α-amylase activity.

“Water hardness” is a measure of the minerals (e.g., calcium andmagnesium) present in water.

“Gelatinization” refers to solubilization of a starch molecule bycooking to form a viscous suspension.

As used herein, the term “liquefaction” or “liquefy” means a process bywhich starch is converted to lower molecular weight (i.e., shorter)dextrins, which are generally more soluble and less viscous than thestarting starch material. This process involves gelatinization of starchsimultaneously with, or followed by, the addition of AmyE or a variantthereof.

As used herein, the term “primary liquefaction” refers to a step ofliquefaction when the slurry's temperature is raised to or near itsgelatinization temperature. Subsequent to the raising of thetemperature, the slurry is sent through a heat exchanger or jet totemperatures from about 90-150° C., e.g., 100-110° C. (200-300° F.,e.g., 220-235° F.). Subsequent to application to a heat exchange or jettemperature, the slurry is held for a period of 3-10 minutes at thattemperature. This step of holding the slurry at 90-150° C. (200-300° F.)is termed primary liquefaction.

As used herein, the term “secondary liquefaction” refers theliquefaction step subsequent to primary liquefaction (heating to 90-150°C. (200-300° F.)), when the slurry is allowed to cool to roomtemperature. This cooling step can be 30 minutes to 180 minutes, e.g. 90minutes to 120 minutes.

As used herein, the term “minutes of secondary liquefaction” refers tothe time that has elapsed from the start of secondary liquefaction, tothe time that the DE is measured.

“Saccharification” refers generally to the enzymatic conversion ofmaltodextrans formed after liquefaction to glucose.

The term “degree of polymerization (DP)” refers to the number (n) ofanhydroglucopyranose units in a given saccharide. Examples of DP1 arethe monosaccharides, such as glucose and fructose. Examples of DP2 arethe disaccharides, such as maltose and sucrose. A DP>3 denotes polymerswith a degree of polymerization of greater than 3.

With respect to starch conversion, the terms “end-product” or “desiredend-product” refer to specified carbon-source-derived molecules, whichare enzymatically converted from a starch substrate.

As used herein, the term “dry solids content (ds)” refers to the totalsolids in a slurry, expressed in % dry weight.

The term “slurry” refers to an aqueous mixture containing insolublesolids.

The term “residual starch” refers to the remaining starch (soluble orinsoluble) in a composition after fermentation or enzymatic hydrolysisof a starch containing substrate.

As used herein “a recycling step” refers to the recycling of mashcomponents, which may include residual starch, enzymes and/ormicroorganisms to ferment substrates comprising starch.

The term “mash” refers to an aqueous mixture including a fermentablecarbon source (e.g., carbohydrate), which may be used to produce afermented product, such as an alcohol. The terms “beer” and “mash” maybe used interchangeability.

The term “stillage” refers to a mixture of non-fermented solids andwater, which represents the residue following removal of alcohol from afermented mash.

The terms “distillers dried grain (DDG)” and “distillers dried grainwith solubles (DDGS)” refer to a useful by-product of grainfermentation.

As used herein “ethanologenic microorganism” refers to a microorganismwith the ability to convert a sugar or oligosaccharide to ethanol. Theethanologenic microorganisms are ethanologenic by virtue of theirability to express one or more enzymes that individually or togetherconvert sugar to ethanol.

As used herein the term “ethanol producer” or ethanol producingmicroorganism” refers to any organism or cell that is capable ofproducing ethanol from a hexose or pentose. Generally, ethanol-producingcells contain an alcohol dehydrogenase and a pyruvate decarboxylase.Examples of ethanol producing microorganisms include fungalmicroorganisms such as yeast. A preferred yeast includes strains ofSacchromyces, particularly, S. cerevisiae.

With respect to amylase enzymes and their substrates, the term“contacting” refers to the placing of the enzyme in sufficiently closeproximity to the substrate to enable the enzyme to convert the substrateto an end-product. Contacting may include mixing.

The term “derived from” means “originated from,” “based on,” “obtainedfrom,” “obtainable from,” or “isolated from,” depending on context.

The term “enzymatic conversion” generally refers to the modification ofa substrate (e.g., starch) by enzyme action (e.g., amylase).

As used herein, the term “disintegration” refers to the hydrolysis ofpolysaccharides in a biofilm matrix connecting and binding togetherindividual microbial cells in the biofilm, whereby the microbial cellscan be released and removed from the biofilm.

A “swatch” is a piece of material, such as a fabric, to which a stainmay be applied for evaluating the cleaning efficiency of a composition.

As used herein the term “specific activity” refers to the number ofmoles of substrate converted to product by an enzyme preparation perunit time under specific conditions. Specific activity is expressed asunits (U)/mg of protein.

As used herein, the term “biologically active” refers to a molecule thatexhibits a preselected biological function.

The term “yield” refers to the amount of end-product produced by aprocess, e.g., expressed in concentration, volume, amount, or apercentage of staring material.

“ATCC” refers to American Type Culture Collection located at Manassas,Va. 20108 (ATCC).

As used herein, a “precipitation agent,” for purposes of purification,refers to a compound effective to precipitate a polypeptide, such asAmyE or a variant thereof, from solution. The form of the precipitatemay be, e.g., crystalline, amorphous, or a blend, thereof.

“NRRL” refers to the Agricultural Research Service Culture Collection,National Center for Agricultural Utilization Research (and previouslyknown as USDA Northern Regional Research Laboratory), Peoria, Ill.

As used herein, a “swatch” is a piece of material, such as a fabric, towhich a stain may be applied, or which has a stain applied. The materialcan be, for example, fabrics made of cotton, polyester or mixtures ofnatural and synthetic fibers. Alternatively, the material can be paper,such as filter paper or nitrocellulose, or a piece of a hard material,such as ceramic, metal, or glass. Exemplary stains include blood, milk,ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay,pigment, oil, or combinations, thereof.

As used herein, a “smaller swatch” is a piece of the swatch that hasbeen cut with a single hole punch device, or a custom manufactured96-hole punch device, or equivalent, where the pattern of the multi-holepunch is matched to standard 96-well microtiter plates, or has beenotherwise removed from the swatch. The swatch can be of textile, paper,metal, or other suitable material. The smaller swatch can have the stainaffixed either before or after it is placed into the well of a 24-, 48-or 96-well microtiter plate.

As used herein, the term “food” encompasses both prepared food andingredients for a food, such as flour, which are capable of providing abenefit to a food preparer of consumer. Food ingredients includesformulations that can be added to a food or foodstuff for the purposesof, e.g., acidifying or emulsifying. The food ingredient may be in theform of a solution or a solid, depending on the use and/or the mode ofapplication and/or the mode of administration.

As used herein, the term “flour” refers to milled or ground cereal grainor Sago or tuber products that have been ground or mashed. In someembodiments, flour may also contain components in addition to the milledor mashed cereal or plant matter, such as a leavening agent. Cerealgrains include wheat, oat, rye, and barley. Tuber products includetapioca flour, cassava flour, and custard powder. Flour also includesground corn flour, maize-meal, rice flour, whole-meal flour, self-risingflour, tapioca flour, cassava flour, ground rice, enriched flower, andcustard powder.

As used herein, the term “stock” refers to grains and plant componentsthat are crushed or broken. For example, barley used in beer productionis a grain that has been coarsely ground or crushed to yield aconsistency appropriate for producing a mash for fermentation. A stockmay include any of the aforementioned types of plants and grains incrushed or coarsely ground forms.

As used herein, the term “performance index (PI)” refers to the ratio ofperformance of a variant polypeptide to a parent polypeptide for aspecified performance characteristic. Within this context, “upmutations” refer to mutations that have a PI>1; “neutral mutations”refer to mutations that have a PI>0.5; “non-deleterious mutations” referto mutations that have a PI>0.05; and “deleterious mutations” refer tomutations that have a PI≦0.05.

As used herein, the terms “added (or additional) glucoamylase (orglucoamylase polypeptide)” or additional polypeptide having glucoamylaseactivity” refers to a glucoamylase enzyme that is not the samepolypeptides as AmyE.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“an enzyme” includes a plurality of such enzymes and reference to “theformulation” includes reference to one or more formulations andequivalents thereof known to those skilled in the art, and so forth.

Numeric ranges are inclusive of the numbers defining the range. Headingsare descriptive and are not intended as limitations. All reference citedherein are incorporated by reference.

1.2. Abbreviations

-   -   The following abbreviations apply unless indicated otherwise:    -   AE alcohol ethoxylate    -   AEO alcohol ethoxylate    -   AEOS alcohol ethoxysulfate    -   AES alcohol ethoxysulfate    -   AGU glucoamylase activity unit    -   AkAA Aspergillus kawachii α-amylase    -   AmyE Bacillus subtilis α-amylase    -   AmyS Geobacillus stearothermophilus α-amylase    -   AS alcohol sulfate    -   BAA bacterial α-amylase    -   cDNA complementary DNA    -   CMC carboxymethylcellulose    -   DE Dextrose Equivalent    -   DI distilled, deionized    -   DNA deoxyribonucleic acid    -   DP3 degree of polymerization with three subunits    -   DPn degree of polymerization with n subunits    -   DS or ds dry solid    -   DTMPA diethyltriaminepentaacetic acid    -   EC enzyme commission for enzyme classification    -   EDTA ethylenediaminetetraacetic acid    -   EDTMPA ethylenediaminetetramethylene phosphonic acid    -   EO ethylene oxide    -   F&HC fabric and household care    -   GAU glucoamylase units    -   HFCS high fructose corn syrup    -   HFSS high fructose starch based syrup    -   IPTG isopropyl β-D-thiogalactoside    -   LA Luria agar    -   LB Luria broth    -   LU Lipase Units    -   L1T leucine (L) residue at position 1 is replaced with a        threonine (T) residue, where amino acids are designated by        single letter abbreviations commonly known in the art    -   MW molecular weight    -   NCBI National Center for Biotechnology Information    -   nm nanometer    -   NOBS nonanoyloxybenzenesulfonate    -   NTA nitrilotriacetic acid    -   OD optical density    -   PCR polymerase chain reaction    -   PEG polyethylene glycol    -   pI isoelectric point    -   ppm parts per million    -   PVA poly(vinyl alcohol)    -   PVP poly(vinylpyrrolidone)    -   RAU Reference Amylase Units    -   RNA ribonucleic acid    -   SAS secondary alkane sulfonates    -   1×SSC 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0    -   SSF simultaneous saccharification and fermentation    -   SSU soluble starch unit, equivalent to the reducing power of 1        mg of glucose released per minute    -   TAED tetraacetylethylenediamine    -   TNBS trinitrobenzenesulfonic acid    -   TrGA Trichoderma reesei glucoamylase    -   w/v weight/volume    -   w/w weight/weight    -   wt wild-type    -   μL microliter    -   μNm microNewton×meter        2. AmyE Polypeptides

2.1. Parental AmyE Polypeptides

AmyE α-amylase refers to a naturally occurring α-amylase (EC 3.2.1.1;1,4-α-D-glucan glucanohydrolase) from B. subtilis, as exemplified by SEQID NO: 1. Related polypeptides have amino acid sequences that differfrom the sequence of a naturally occurring AmyE, for example, amino acidsequences that have at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or even at least about 99% sequence identity with SEQ ID NO:1, as measured with the BLAST sequence alignment algorithm with defaultmatching parameters.

Another exemplary AmyE polypeptide is Amy31A having the amino acidsequence of SEQ ID NO: 3 (Amy31A). Amy31A is described in Ohdan et al.,“Characteristics of two forms of alpha-amylases and structuralimplication,” Appl. Environ. Microbiol. 65(10): 4652-58 (1999) andUniProtKB/TrEMBL Accession No. O82953 (SEQ ID NO: 3). Amy31A has about86% sequence identity to the AmyE of SEQ ID NO: 1, using the BLASTalgorithm. Additional AmyE polypeptides include, but are not limited to,the AmyE having the amino acid sequence described in NCBI Accession Nos.ABW75769, ABK54355, AAF14358, AAT01440, AAZ30064, NP_(—)388186,AAQ83841, and BAA31528, the contents of which are incorporated here byreference.

The representative AmyE amino acid sequence set forth in SEQ ID NO: 1 isthat of a mature form, which lacks the native signal sequence. Themature form of an AmyE is referred to elsewhere as “AmyE full-length.”Generally, the mature form of AmyE is of the most interest as an enzyme,although it may be desirable to express the immature form (with a signalsequence) to affect secretion from a host cell. The native signalsequence of this AmyE is 41 amino acid residues in length and is shownas SEQ ID NO: 9. The N-terminal 45 amino acid residues of SEQ ID NO: 3are the signal sequence of Amy31A. A sequence alignment between AmyE(SEQ ID NO: 1) and Amy31A (without the signal sequence) is depicted inFIG. 1.

AmyE polypeptides may have a deletion of the C-terminal starch bindingdomain, as exemplified by the truncated AmyE polypeptide having theamino acid sequence of SEQ ID NO: 2 (AmyE-tr). This polypeptide istruncated from residue D425 (referring to SEQ ID NO: 1). A 2.5 Åresolution crystal structure of AmyE-tr is available at Protein DatabankAccession No. 1BAG, which is disclosed in Fujimoto et al. (1998)“Crystal structure of a catalytic-site mutant alpha-amylase from B.subtilis complexed with maltopentaose,” J. Mol. Biol. 277:393-407. AmyEmay be truncated at other positions, e.g., Y423, P424, D426 or I427 ofthe AmyE of SEQ ID NO: 1, provided all or part of the C-terminal starchbinding domain is removed. Similar truncations can be made to Amy31A andother AmyE polypeptides.

2.2. AmyE Variants

AmyE variants comprise at least one amino acid modification compared tothe naturally-occurring AmyE of SEQ ID NO: 1, or compared to SEQ ID NO:2 (the truncated polypeptide). Accordingly, the AmyE polypeptides of SEQID NO: 1 or SEQ ID NO: 2 may be referred to as “parental polypeptides,”“parental enzymes,” or “parental sequences,” from which AmyE variantsare derived. The amino acid residues that are not modified (i.e., theremaining contiguous amino acid sequences) may be identical to those ofSEQ ID NOs: 1 or 2, identical to those of SEQ ID NO: 3, or identical tothose of NCBI Accession Nos. ABW75769, ABK54355, AAF14358, AAT01440,AAZ30064, NP_(—)388186, AAQ83841, and BAA31528. Alternatively, theremaining amino acid sequences may have a specified degree of sequenceidentity to one or more of these sequences as measured using, e.g., aBLAST alignment of the protein sequences with default alignmentparameters. For example the remaining sequences may have at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or even at least about 99%sequence identity with the AmyE of SEQ ID NO: 1 or SEQ ID NO: 2.

AmyE variants may have a single amino acid modification or may have,e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more amino acidmodifications compared to the amino acid sequence of SEQ ID NO: 1 or SEQID NO: 2. Modifications include substitutions, insertions, deletions, orcombinations, thereof. In some cases, the modifications are to aminoacid residues that are not required for biological function. Theselection of amino acid residues to be modified may be guided bysequence homology among AmyE sequences. Generally, amino acids that arewell conserved in AmyE sequences are more likely to be required forbiological activity. Conversely, amino acid positions that vary amongAmyE sequences are less likely to be required for biological activity.For example, amino acid residues that differ in the alignment betweenAmyE and Amy31A, shown in bold font in FIG. 1, likely can be modified inan AmyE variant without loss of biological activity.

Preferred AmyE variants have at least partial 1,4-α-D-glucanglucanohydrolase activity, compared to a naturally-occurring AmyE and atleast one altered property compared to a naturally-occurring AmyE. Thealtered property may be with respect to specific activity towardsstarch, maltoheptaose, and/or maltotriose substrates, substratespecificity, thermostability, temperature optima, pH optima, pH and/ortemperature range, oxidative stability, ability to reduce the viscosityof a starch composition, or the like. In some cases, the alteredproperty of the AmyE variant relates to the specific activity on aparticular corn flour, maltotriose, maltoheptaose substrate atparticular pH (e.g., 4 or 5.8), heat stability at a particulartemperature, (e.g., 60° C.), or cleaning performance at a particular pH(e.g., 8 or pH 10). The altered property may be characterized by aPerformance Index (PI), where the PI is a ratio of performance of theAmyE variant compared to a wild-type AmyE. In some embodiments, the PIis greater than about 0.5, while in other embodiments, the PI is about 1or is greater than 1.

Specific residues that may be substituted to impart beneficialproperties on a resulting AmyE variant include one or more of thefollowing: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 267, 268, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, and 425. Modifications at any of thesepositions produces a variant polypeptide having a performance index (PI)greater than 0.5 for protein expression, and a PI greater than 1 for atleast one characteristic that improves enzyme performance.

A subset of residues that may be substituted to impart beneficialproperties on a resulting AmyE variant include one or more of thefollowing: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 63, 64, 65, 66, 67, 68, 69, 72, 73, 74, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99,100, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 118, 119, 121, 124, 125, 126, 128, 129, 130, 131, 132, 134, 135,136, 140, 141, 142, 143, 144, 147, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 170, 171, 172,175, 179, 180, 181, 184, 186, 187, 188, 189, 190, 192, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 207, 209, 211, 212, 213, 214,217, 218, 219, 221, 222, 223, 224, 225, 226, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 267, 268, 270, 271, 272, 273, 274, 275, 276, 277, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,294, 295, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 324,325, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, and425. Modifications at any of these positions produces a variantpolypeptide having a performance index (PI) greater than 0.5 for proteinexpression, and a PI greater than 1.1 for at least one characteristicthat improves enzyme performance.

In some case, one or more positions are selected from the groupconsisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 98, 99, 100, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 124, 125,126, 127, 128, 129, 130, 131, 132, 134, 135, 136, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,and 425, which positions are non-fully restrictive for performance ineither the full-length or truncated parental polypeptide.

Some positions were determined to be non-fully restrictive forperformance in the context of the truncated AmyE parental polypeptide(i.e., 1, 2, 3, 4, 5, 8, 18, 20, 23, 24, 25, 27, 28, 30, 35, 44, 45, 47,49, 50, 51, 52, 54, 56, 59, 68, 73, 76, 78, 85, 88, 89, 90, 91, 106,107, 108, 109, 112, 115, 116, 118, 119, 124, 125, 126, 127, 131, 132,134, 142, 143, 152, 153, 156, 160, 163, 166, 167, 184, 185, 187, 188,190, 192, 195, 199, 200, 201, 202, 203, 212, 213, 214, 218, 219, 221,222, 223, 233, 234, 238, 240, 241, 243, 245, 247, 248, 250, 251, 252,253, 254, 255, 257, 259, 260, 274, 275, 276, 277, 282, 283, 284, 287,307, 308, 309, 310, 311, 312, 313, 314, 317, 318, 319, 320, 321, 323,324, 325, 327, 328, 331, 333, 344, 346, 347, 349, 357, 358, 359, 367,368, 369, 378, 380, 382, 385, 386, 388, 390, 393, 395, 400, 401, 402,and 406), and some positions were determined to be non-fully restrictivefor performance in the context of the full-length AmyE parentalpolypeptide (i.e., 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 21, 22,26, 27, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46,48, 52, 53, 55, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72,74, 77, 79, 80, 81, 82, 83, 84, 86, 87, 88, 89, 92, 93, 94, 95, 96, 98,99, 100, 103, 104, 105, 110, 111, 113, 114, 117, 121, 122, 126, 128,129, 130, 131, 135, 136, 138, 139, 140, 141, 144, 145, 146, 147, 148,149, 150, 151, 154, 155, 157, 158, 159, 161, 162, 164, 165, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 183,184, 186, 189, 191, 193, 194, 196, 197, 198, 204, 205, 206, 207, 208,209, 210, 211, 215, 216, 217, 220, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 235, 236, 237, 238, 239, 241, 242, 244, 246, 249, 256,258, 260, 261, 262, 263, 264, 265, 267, 268, 269, 270, 271, 272, 273,278, 279, 280, 281, 285, 286, 288, 289, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 312, 315, 316,322, 326, 329, 330, 332, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 348, 350, 351, 352, 353, 354, 355, 356, 360, 361, 362,363, 364, 365, 366, 370, 371, 372, 373, 374, 375, 376, 377, 379, 380,381, 383, 384, 387, 389, 391, 392, 394, 396, 397, 398, 399, 402, 403,404, 405, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, 425).

In some cases, the modification is a substitution of one or more aminoresidues present in the parental polypeptide to different amino acidresidues, for example: 1A, 1C, 1D, 1E, 1F, 1G, 1H, 1K, 1M, 1N, 1Q, 1R,1S, 1T, 1V, 1W, 1Y, 2A, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2K, 2L, 2M, 2N, 2P,2Q, 2R, 2S, 2V, 2W, 2Y, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3K, 3L, 3M, 3N, 3P,3Q, 3R, 3S, 3V, 3W, 3Y, 4C, 4D, 4E, 4F, 4G, 4H, 4I, 4K, 4L, 4M, 4N, 4Q,4S, 4T, 4V, 4W, 4Y, 5A, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5K, 5L, 5N, 5R, 5V,5W, 5Y, 6C, 6D, 6E, 6H, 6K, 6L, 6M, 6N, 6P, 6Q, 6R, 6S, 6T, 6V, 6W, 7A,7C, 7D, 7E, 7F, 7G, 7H, 7I, 7L, 7M, 7N, 7P, 7Q, 7R, 7S, 7T, 7W, 7Y, 8A,8C, 8E, 8F, 8G, 8H, 8I, 8K, 8L, 8M, 8N, 8P, 8Q, 8R, 8T, 8V, 8W, 8Y, 9A,9C, 9D, 9E, 9F, 9H, 9I, 9K, 9M, 9N, 9P, 9R, 9S, 9T, 9V, 9W, 9Y, 10A,10I, 10L, 10M, 10N, 10P, 10Q, 10S, 10y, 11A, 11F, 11G, 11H, 11M, 11S,11V, 11W, 11Y, 12I, 12M, 12V, 13A, 13C, 13D, 13E, 13F, 13G, 13I, 13L,13M, 13Q, 13T, 13V, 13W, 13Y, 14C, 14F, 14G, 14M, 14N, 14S, 14T, 14V,15A, 15F, 16A, 16D, 16E, 16F, 16G, 16H, 16I, 16L, 16M, 16Q, 16S, 16T,16V, 17A, 17F, 17I, 17M, 17Q, 17Y, 18A, 18C, 18D, 18E, 18G, 18H, 18M,18N, 18Q, 18R, 18T, 19A, 19C, 19H, 19L, 19M, 19N, 19S, 19W, 19Y, 20A,20C, 20D, 20F, 20G, 20H, 20I, 20K, 20L, 20M, 20P, 20Q, 20R, 20S, 20T,20V, 20W, 20Y, 21A, 21C, 21D, 21E, 21H, 21I, 21K, 21L, 21M, 21N, 21Q,21R, 21S, 21V, 22I, 22M, 22Q, 22S, 22T, 22V, 23A, 23C, 23D, 23E, 23F,23G, 23H, 23I, 23L, 23M, 23N, 23R, 23S, 23T, 23V, 23W, 23Y, 24A, 24C,24D, 24F, 24G, 24L, 24N, 24P, 24Q, 24R, 24S, 24T, 24V, 24Y, 25A, 25C,25D, 25E, 25F, 25G, 25H, 25I, 25K, 25L, 25R, 25S, 25T, 25V, 25W, 25Y,26A, 26F, 26I, 26L, 26V, 27A, 27C, 27D, 27E, 27F, 27G, 27H, 27I, 27L,27M, 27N, 27P, 27Q, 27R, 27S, 27T, 27V, 27W, 27Y, 28A, 28C, 28F, 28G,28H, 28I, 28K, 28L, 28M, 28N, 28P, 28Q, 28R, 28S, 28T, 28V, 28W, 28Y,29A, 29C, 29F, 29L, 29M, 29T, 29V, 30A, 30C, 30D, 30E, 30F, 30G, 30I,30K, 30L, 30M, 30N, 30P, 30Q, 30R, 30S, 30T, 30V, 30W, 30Y, 31A, 31C,31E, 31F, 31G, 31H, 31I, 31K, 31L, 31M, 31N, 31Q, 31S, 31T, 31V, 31W,31Y, 32D, 32F, 32G, 32H, 32K, 32L, 32M, 32Q, 32S, 32T, 32V, 32Y, 33A,33C, 33D, 33E, 33F, 33H, 33I, 33K, 33L, 33M, 33P, 33Q, 33S, 33T, 33W,33Y, 34A, 34F, 34I, 34P, 34W, 35A, 35C, 35F, 35G, 35H, 35I, 35L, 35M,35N, 35P, 35Q, 35R, 35S, 35V, 35W, 35Y, 36C, 36D, 36E, 36F, 36H, 36I,36K, 36L, 36M, 36N, 36Q, 36R, 36S, 36T, 36Y, 37L, 37M, 37N, 37V, 38A,38C, 38D, 38E, 38H, 38L, 38M, 38N, 38P, 38V, 39A, 39C, 39I, 39L, 39M,39N, 39P, 39S, 39V, 40A, 40D, 40M, 40N, 40P, 40Q, 40T, 40V, 40W, 41A,41C, 41E, 41G, 41N, 41S, 41V, 42A, 42L, 42M, 42P, 42V, 43A, 43G, 43H,43L, 43M, 43Q, 43S, 43T, 43V, 44A, 44C, 44D, 44E, 44F, 44G, 44H, 44I,44K, 44L, 44M, 44N, 44P, 44R, 44S, 44T, 44V, 44W, 44Y, 45A, 45C, 45F,45G, 45H, 45I, 45L, 45M, 45N, 45P, 45Q, 45S, 45T, 45Y, 46A, 46C, 46D,46E, 46F, 46H, 46I, 46L, 46M, 46N, 46Q, 46R, 46S, 46T, 46V, 46W, 46Y,47A, 47C, 47D, 47F, 47G, 47H, 47I, 47K, 47L, 47N, 47P, 47R, 47S, 47T,47V, 47Y, 48A, 48C, 48D, 48E, 48F, 48H, 48I, 48K, 48L, 48N, 48P, 48S,48T, 48V, 48W, 49A, 49C, 49D, 49F, 49G, 49H, 49I, 49K, 49L, 49P, 49Q,49R, 49S, 49T, 49V, 49W, 49Y, 50A, 50C, 50E, 50F, 50G, 50H, 50I, 50K,50L, 50M, 50N, 50P, 50R, 50S, 50T, 50V, 50W, 50Y, 51A, 51C, 51D, 51E,51F, 51H, 51I, 51K, 51L, 51M, 51N, 51P, 51Q, 51R, 51S, 51T, 51V, 51W,52A, 52C, 52E, 52F, 52G, 52H, 52I, 52K, 52L, 52M, 52N, 52P, 52Q, 52R,52S, 52T, 52V, 52W, 52Y, 53A, 53C, 53E, 53F, 53G, 53H, 53I, 53L, 53N,53P, 53R, 53S, 53T, 53V, 53W, 53Y, 54A, 54C, 54D, 54F, 54G, 54H, 54I,54L, 54M, 54N, 54P, 54Q, 54R, 54T, 54V, 54W, 54Y, 55A, 55C, 55D, 55E,55F, 55G, 55H, 55I, 55N, 55P, 55Q, 55S, 55T, 55Y, 56A, 56D, 56E, 56F,56G, 56I, 56K, 56L, 56M, 56N, 56P, 56Q, 56R, 56T, 56V, 56W, 56Y, 57A,57C, 57D, 57E, 57F, 57H, 57I, 57K, 57L, 57M, 57Q, 57R, 57S, 57T, 57V,57W, 57Y, 58A, 58F, 58H, 59A, 59C, 59D, 59E, 59F, 59G, 59H, 59K, 59L,59N, 59P, 59R, 59S, 59T, 59V, 59W, 60A, 60C, 60D, 60E, 60G, 60I, 60K,60L, 60M, 60N, 60Q, 60R, 60T, 60V, 61C, 61D, 61E, 61F, 61M, 61S, 61T,61V, 62A, 62C, 62D, 62F, 62G, 62H, 62I, 62K, 62L, 62Q, 62S, 62T, 62V,63A, 63C, 63D, 63F, 63G, 63H, 63K, 63M, 63N, 63R, 63S, 64A, 64G, 64H,64I, 64L, 64M, 64N, 64S, 64V, 64Y, 65A, 65C, 65E, 65H, 65I, 65K, 65L,65M, 65Q, 65R, 65S, 66D, 66E, 66F, 66G, 66H, 66I, 66K, 66L, 66M, 66N,66Q, 66R, 66T, 66V, 66W, 66Y, 67A, 67C, 67D, 67E, 67F, 67G, 67I, 67K,67L, 67N, 67P, 67Q, 67S, 67T, 67W, 68A, 68C, 68D, 68E, 68F, 68G, 68H,68I, 68L, 68M, 68N, 68P, 68R, 68S, 68T, 68V, 68W, 68Y, 69A, 69C, 69M,69P, 69T, 69V, 70A, 70E, 70H, 70N, 70S, 71S, 72C, 72D, 72E, 72F, 72G,72H, 72I, 72K, 72L, 72P, 72Q, 72S, 72T, 72V, 72W, 72Y, 73A, 73C, 73E,73F, 73H, 73I, 73K, 73L, 73M, 73P, 73S, 73T, 73V, 73W, 74A, 74E, 74F,74M, 74S, 74T, 74Y, 75A, 75C, 75D, 75E, 75P, 76A, 76D, 76E, 76F, 76G,76I, 76L, 76M, 76P, 76Q, 76R, 76S, 76V, 76Y, 77A, 77C, 77D, 77G, 77H,77I, 77K, 77L, 77R, 77S, 77T, 77V, 77W, 77Y, 78A, 78C, 78D, 78E, 78F,78G, 78H, 78I, 78K, 78L, 78M, 78N, 78P, 78R, 78S, 78T, 78V, 78W, 78Y,79A, 79G, 79L, 79M, 79N, 79Q, 79S, 79T, 80A, 80L, 80M, 80W, 80Y, 81A,81C, 81D, 81E, 81G, 81H, 81I, 81L, 81M, 81N, 81Q, 81R, 81S, 81T, 81V,81W, 81Y, 82A, 82D, 82F, 82G, 82I, 82K, 82L, 82M, 82Q, 82R, 82S, 82T,82V, 82W, 82Y, 83A, 83F, 83I, 83L, 83T, 83V, 84A, 84D, 84E, 84G, 84I,84K, 84M, 84N, 84Q, 84S, 84T, 84V, 85D, 85E, 85F, 85G, 85I, 85K, 85L,85M, 85N, 85R, 85S, 85T, 85V, 85W, 86C, 86D, 86E, 86F, 86G, 86I, 86K,86L, 86M, 86N, 86Q, 86R, 86S, 86V, 86W, 86Y, 87F, 87G, 87T, 88A, 88C,88D, 88F, 88G, 88H, 88I, 88K, 88L, 88M, 88N, 88Q, 88R, 88S, 88T, 88V,88W, 88Y, 89A, 89C, 89D, 89F, 89G, 89H, 89I, 89K, 89L, 89M, 89N, 89P,89Q, 89R, 89S, 89T, 89V, 89W, 89Y, 90C, 90D, 90E, 90F, 90G, 90H, 90I,90K, 90L, 90M, 90N, 90Q, 90R, 90S, 90T, 90V, 90W, 91A, 91C, 91D, 91E,91F, 91H, 91K, 91L, 91M, 91N, 91P, 91Q, 91R, 91S, 91T, 91W, 91Y, 92L,92N, 92T, 92V, 93A, 93C, 93D, 93E, 93F, 93G, 93I, 93L, 93M, 93N, 93P,93Q, 93R, 93S, 93T, 93V, 93W, 93Y, 94A, 94C, 94I, 94M, 94T, 95A, 95F,95L, 95M, 95V, 95Y, 96A, 96C, 96I, 96L, 96M, 96P, 96T, 97A, 97E, 97M,97W, 98C, 98G, 98I, 98L, 98M, 98T, 98V, 99A, 99C, 99E, 99F, 99G, 99I,99L, 99M, 99N, 99P, 99S, 99T, 100A, 100C, 100F, 100M, 100N, 100P, 100T,100V, 100Y, 101A, 102A, 102G, 102Q, 102S, 102W, 102Y, 103A, 103C, 103I,103M, 103N, 103S, 103V, 104A, 104C, 104S, 105C, 105D, 105E, 105F, 105G,105H, 105K, 105L, 105M, 105Q, 105R, 105T, 105V, 105W, 105Y, 106A, 106C,106E, 106F, 106H, 106I, 106K, 106L, 106M, 106N, 106Q, 106R, 106S, 106T,106V, 106W, 106Y, 107A, 107C, 107E, 107F, 107G, 107H, 107I, 107K, 107L,107M, 107N, 107P, 107Q, 107R, 107S, 107T, 107V, 107W, 108C, 108D, 108E,108F, 108G, 108H, 108I, 108K, 108L, 108N, 108P, 108R, 108S, 108T, 108V,108W, 108Y, 109C, 109D, 109E, 109F, 109G, 109H, 109I, 109K, 109L, 109M,109N, 109P, 109R, 109S, 109T, 109V, 109W, 109Y, 110L, 110M, 110V, 111A,111C, 111E, 111F, 111G, 111H, 111I, 111K, 111L, 111M, 111N, 111P, 111Q,111R, 111T, 111V, 111W, 111Y, 112A, 112C, 112D, 112E, 112F, 112G, 112H,112I, 112K, 112L, 112M, 112P, 112Q, 112R, 112S, 112T, 112V, 112W, 112Y,113A, 113D, 113F, 113G, 113I, 113K, 113L, 113M, 113N, 113P, 113Q, 113S,113T, 113V, 113W, 113Y, 114F, 114L, 114P, 114T, 115A, 115C, 115F, 115G,115H, 115I, 115L, 115M, 115N, 115Q, 115R, 115S, 115T, 115V, 115W, 115Y,116A, 116D, 116E, 116F, 116G, 116H, 116I, 116L, 116N, 116Q, 116R, 116T,116V, 116W, 116Y, 117F, 117L, 117N, 117Q, 117V, 117W, 117Y, 118A, 118C,118D, 118E, 118F, 118G, 118H, 118I, 118K, 118L, 118M, 118N, 118Q, 118R,118S, 118T, 118V, 118W, 118Y, 119A, 119C, 119D, 119E, 119F, 119G, 119H,119I, 119K, 119L, 119M, 119Q, 119R, 119S, 119T, 119V, 119Y, 121A, 121M,121N, 121S, 122R, 123E, 124A, 124C, 124D, 124E, 124F, 124G, 124I, 124K,124M, 124Q, 124R, 124S, 124T, 124V, 124Y, 125A, 125D, 125E, 125F, 125G,125H, 125I, 125K, 125L, 125M, 125N, 125P, 125Q, 125R, 125S, 125V, 125W,125Y, 126A, 126C, 126D, 126F, 126G, 126H, 126I, 126K, 126L, 126N, 126P,126R, 126S, 126T, 126V, 126W, 126Y, 127C, 127L, 127M, 127V, 128A, 128C,128D, 128E, 128F, 128G, 128H, 128I, 128L, 128M, 128N, 128Q, 128R, 128S,128T, 128V, 128Y, 129A, 129C, 129D, 129E, 129F, 129H, 129I, 129K, 129L,129M, 129R, 129S, 129T, 129V, 129Y, 130A, 130C, 130D, 130F, 130G, 130H,130I, 130K, 130L, 130M, 130P, 130R, 130T, 130V, 130Y, 131A, 131C, 131D,131E, 131F, 131G, 131H, 131I, 131K, 131L, 131M, 131N, 131Q, 131R, 131T,131V, 131W, 131Y, 132A, 132C, 132E, 132H, 132I, 132L, 132M, 132N, 132Q,132S, 132W, 132Y, 134C, 134D, 134E, 134F, 134G, 134I, 134L, 134M, 134N,134R, 134S, 134T, 134V, 134Y, 135A, 135C, 135E, 135M, 135N, 135Q, 135R,136A, 136C, 136F, 136L, 136T, 136Y, 137C, 138A, 138C, 138F, 138H, 138N,138R, 138W, 138Y, 139A, 139C, 139G, 139H, 139L, 139M, 139S, 140A, 140C,140F, 140G, 141A, 141F, 141H, 141P, 141Q, 141S, 141T, 141V, 141Y, 142C,142D, 142F, 142G, 142H, 142I, 142K, 142M, 142Q, 142R, 142S, 142T, 142W,142Y, 143A, 143C, 143D, 143F, 143K, 143L, 143M, 143N, 143Q, 143R, 143S,143W, 144G, 144S, 144T, 144V, 144W, 145W, 146A, 146E, 146M, 146T, 147C,147F, 147H, 147I, 147L, 147N, 147P, 147Y, 148A, 148C, 148F, 148H, 148K,148M, 148R, 148Y, 149S, 150A, 150H, 150N, 150S, 151A, 151C, 151D, 151E,151F, 151G, 151H, 151I, 151K, 151L, 151M, 151Q, 151R, 151S, 151T, 151V,151Y, 152A, 152C, 152D, 152E, 152F, 152G, 152H, 152I, 152K, 152L, 152M,152N, 152P, 152Q, 152R, 152S, 152V, 152W, 152Y, 153A, 153C, 153D, 153E,153F, 153G, 153H, 153I, 153K, 153L, 153M, 153N, 153P, 153R, 153S, 153T,153V, 153W, 153Y, 154A, 154C, 154I, 154N, 154P, 154Q, 154S, 154T, 154Y,155A, 155C, 155E, 155F, 155G, 155H, 155I, 155L, 155M, 155T, 155V, 155W,156A, 156C, 156D, 156E, 156F, 156G, 156H, 156I, 156K, 156L, 156N, 156Q,156R, 156T, 156V, 156W, 156Y, 157A, 157C, 157F, 157H, 157I, 157M, 157T,157V, 158A, 158F, 158H, 158I, 158M, 158Q, 158S, 158T, 158V, 159A, 159C,159E, 159F, 159G, 159H, 159I, 159L, 159M, 159N, 159R, 159S, 159T, 159V,159W, 159Y, 160A, 160C, 160D, 160E, 160F, 160G, 160H, 160I, 160K, 160L,160M, 160N, 160Q, 160S, 160T, 160V, 160W, 160Y, 161A, 161C, 161G, 161H,161K, 161L, 161M, 161N, 161S, 162A, 162C, 162E, 162F, 162I, 162M, 162V,163A, 163C, 163E, 163F, 163G, 163H, 163I, 163K, 163L, 163N, 163Q, 163R,163S, 163T, 163V, 163W, 163Y, 164A, 164F, 164G, 164H, 164I, 164L, 164M,164N, 164Q, 164S, 164T, 164V, 164W, 164Y, 165C, 165G, 165I, 165L, 165M,165Q, 165S, 165T, 165V, 165W, 165Y, 166A, 166C, 166D, 166E, 166F, 166G,166H, 166I, 166K, 166M, 166N, 166Q, 166R, 166S, 166T, 166V, 166W, 166Y,167A, 167C, 167D, 167E, 167F, 167G, 167H, 167I, 167K, 167L, 167M, 167Q,167R, 167S, 167T, 167V, 167W, 167Y, 168C, 168E, 168F, 168G, 168I, 168K,168L, 168M, 168N, 168S, 168T, 168V, 168W, 168Y, 169L, 170C, 170G, 170V,171A, 171C, 171E, 171F, 171G, 171H, 171I, 171L, 171M, 171N, 171Q, 171R,171V, 172A, 172C, 172E, 172F, 172P, 173I, 173M, 173V, 173Y, 174D, 174E,174G, 174H, 174L, 174Q, 174V, 174Y, 175H, 175M, 175W, 175Y, 176E, 176F,176I, 176K, 176L, 176V, 176Y, 177C, 177G, 177M, 177Q, 177S, 178C, 178G,178M, 178S, 178T, 179A, 179C, 179G, 179H, 179I, 179L, 179M, 179P, 179S,179T, 179V, 179W, 179Y, 180A, 180D, 180M, 180N, 180Y, 181A, 181C, 181L,181M, 181V, 182A, 183C, 183M, 184A, 184C, 184D, 184E, 184F, 184G, 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353Y,354A, 354C, 354M, 354P, 354Q, 354S, 354T, 355C, 355D, 355E, 355F, 355G,355I, 355K, 355L, 355M, 355N, 355T, 355V, 355W, 355Y, 356D, 356E, 356F,356G, 356H, 356I, 356K, 356L, 356M, 356P, 356Q, 356T, 356W, 356Y, 357A,357C, 357D, 357E, 357F, 357H, 357I, 357K, 357L, 357M, 357N, 357P, 357Q,357R, 357S, 357T, 357V, 357W, 357Y, 358A, 358C, 358D, 358E, 358F, 358G,358H, 358I, 358K, 358L, 358M, 358P, 358Q, 358R, 358S, 358T, 358V, 358W,358Y, 359A, 359C, 359D, 359E, 359F, 359G, 359H, 359I, 359K, 359L, 359M,359P, 359Q, 359R, 359S, 359T, 359V, 359W, 359Y, 360F, 360H, 360L, 360N,360P, 360R, 360T, 360W, 361A, 361C, 361G, 361H, 361L, 361M, 361N, 361Q,361S, 361T, 361V, 361W, 361Y, 362A, 362C, 362E, 362H, 362I, 362L, 362M,362Q, 362S, 362T, 362V, 362Y, 363D, 363E, 363F, 363G, 363H, 363N, 363Q,363R, 363S, 363V, 363W, 363Y, 364A, 364C, 364D, 364E, 364G, 364I, 364L,364M, 364Q, 364S, 364T, 364V, 365A, 365C, 365D, 365F, 365G, 365I, 365K,365L, 365M, 365N, 365R, 365S, 365T, 365V, 365W, 365Y, 366A, 366C, 366E,366F, 366G, 366H, 366K, 366L, 366M, 366S, 366T, 366V, 367A, 367C, 367D,367E, 367F, 367H, 367I, 367K, 367L, 367M, 367N, 367P, 367R, 367S, 367T,367V, 367W, 367Y, 368D, 368F, 368G, 368I, 368K, 368L, 368M, 368N, 368P,368Q, 368R, 368T, 368V, 368W, 368Y, 369A, 369C, 369D, 369E, 369F, 369G,369I, 369K, 369L, 369M, 369N, 369P, 369Q, 369R, 369S, 369T, 369V, 369W,369Y, 370A, 371A, 371C, 371F, 371G, 371H, 371I, 371L, 371M, 371N, 371Q,371S, 371T, 371W, 371Y, 372A, 372C, 372G, 372I, 372L, 372M, 372N, 372Q,372S, 372T, 373A, 373C, 373F, 373G, 373I, 373M, 373Q, 373S, 373T, 373V,373W, 373Y, 374C, 374E, 374G, 374I, 374L, 374M, 374N, 374S, 374T, 374V,375A, 375C, 375D, 375F, 375G, 375H, 375L, 375M, 375Q, 375S, 375T, 375V,375W, 375Y, 376C, 376D, 376E, 376F, 376G, 376H, 376I, 376L, 376M, 376N,376P, 376Q, 376S, 376T, 376V, 377F, 377H, 377I, 377K, 377L, 377P, 377T,377W, 377Y, 378A, 378C, 378D, 378E, 378F, 378G, 378H, 378I, 378K, 378L,378M, 378N, 378P, 378Q, 378R, 378T, 378V, 378W, 378Y, 379A, 379D, 379G,379H, 379I, 379K, 379L, 379Q, 379T, 379W, 379Y, 380A, 380C, 380D, 380E,380F, 380G, 380H, 380I, 380L, 380M, 380N, 380P, 380Q, 380R, 380T, 380V,380W, 380Y, 381A, 381G, 381I, 381K, 381N, 381P, 381Q, 381R, 381S, 381T,381W, 381Y, 382A, 382C, 382D, 382F, 382G, 382H, 382I, 382K, 382L, 382M,382N, 382P, 382Q, 382R, 382T, 382V, 382W, 382Y, 383A, 383C, 383E, 383F,383G, 383H, 383L, 383N, 383P, 383Q, 383S, 383T, 383V, 383W, 383Y, 384A,384D, 384F, 384G, 384H, 384I, 384K, 384L, 384P, 384Q, 384S, 384T, 384V,384W, 385A, 385C, 385D, 385E, 385F, 385G, 385H, 385I, 385K, 385L, 385M,385N, 385P, 385Q, 385R, 385S, 385V, 385W, 385Y, 386C, 386D, 386F, 386G,386H, 386I, 386L, 386N, 386P, 386R, 386S, 386T, 386V, 386W, 386Y, 387A,387D, 387E, 387G, 387I, 387L, 387N, 387Q, 387S, 388A, 388C, 388D, 388E,388F, 388G, 388H, 388I, 388L, 388M, 388N, 388P, 388Q, 388R, 388S, 388T,388V, 388W, 388Y, 389C, 389E, 389F, 389H, 389I, 389K, 389M, 389N, 389Q,389S, 389T, 389V, 389W, 389Y, 390A, 390C, 390D, 390E, 390F, 390G, 390H,390I, 390K, 390L, 390M, 390N, 390R, 390S, 390T, 390V, 390W, 390Y, 391E,391F, 391G, 391H, 391I, 391K, 391L, 391N, 391P, 391R, 391S, 391T, 391V,391W, 391Y, 392A, 392C, 392D, 392E, 392F, 392H, 392K, 392L, 392M, 392N,392Q, 392R, 392S, 392V, 392Y, 393A, 393C, 393D, 393F, 393G, 393H, 393I,393L, 393M, 393P, 393Q, 393S, 393T, 393V, 393W, 393Y, 394A, 394C, 394E,394F, 394H, 394I, 394K, 394L, 394M, 394N, 394P, 394Q, 394R, 394S, 394T,394V, 394W, 395A, 395C, 395E, 395F, 395G, 395H, 395I, 395K, 395L, 395M,395N, 395P, 395Q, 395R, 395S, 395T, 395V, 395W, 395Y, 396A, 396C, 396D,396E, 396G, 396M, 396P, 396S, 396T, 397A, 397C, 397D, 397E, 397F, 397G,397H, 397I, 397L, 397M, 397P, 397R, 397S, 397T, 397V, 397W, 398C, 398D,398E, 398F, 398G, 398I, 398L, 398M, 398N, 398P, 398Q, 398R, 398S, 398T,398V, 398W, 398Y, 399A, 399C, 399D, 399E, 399F, 399H, 399I, 399K, 399L,399P, 399R, 399S, 399T, 399V, 399W, 399Y, 400C, 400D, 400E, 400F, 400G,400H, 400I, 400K, 400L, 400M, 400N, 400P, 400Q, 400R, 400S, 400T, 400V,400W, 400Y, 401A, 401C, 401D, 401E, 401F, 401H, 401I, 401K, 401L, 401M,401N, 401P, 401Q, 401R, 401S, 401T, 401V, 401W, 401Y, 402A, 402C, 402D,402E, 402F, 402G, 402H, 402I, 402K, 402L, 402M, 402N, 402P, 402Q, 402R,402T, 402V, 402W, 402Y, 403A, 403C, 403E, 403G, 403H, 403I, 403M, 403N,403Q, 403S, 403T, 403V, 403W, 403Y, 404D, 404E, 404F, 404G, 404H, 404I,404L, 404M, 404N, 404P, 404R, 404T, 404V, 404W, 404Y, 405E, 405F, 405G,405H, 405I, 405K, 405Q, 405S, 405T, 406D, 406F, 406L, 406T, 406Y, 407A,407C, 407E, 407F, 407G, 407H, 407I, 407K, 407M, 407N, 407P, 407Q, 407R,407S, 407T, 407V, 407W, 407Y, 408A, 408D, 408E, 408F, 408H, 408I, 408K,408N, 408P, 408Q, 408S, 408T, 408V, 408Y, 409A, 409C, 409D, 409E, 409F,409H, 409I, 409L, 409M, 409Q, 409R, 409T, 409V, 409W, 409Y, 410F, 410G,410I, 410K, 410Q, 410S, 410T, 410V, 410W, 410Y, 411A, 411D, 411E, 411F,411G, 411H, 411I, 411L, 411M, 411N, 411Q, 411R, 411S, 411V, 411W, 411Y,412A, 412D, 412E, 412H, 412I, 412K, 412L, 412M, 412N, 412R, 412S, 412T,412V, 412Y, 413C, 413E, 413F, 413G, 413I, 413L, 413M, 413N, 413P, 413R,413S, 413V, 413W, 413Y, 414A, 414C, 414E, 414F, 414G, 414H, 414L, 414M,414N, 414P, 414Q, 414T, 414V, 414W, 415D, 415E, 415F, 415G, 415H, 415I,415K, 415P, 415Q, 415R, 415V, 415W, 416F, 416I, 416L, 416P, 416Q, 416R,416T, 416V, 416Y, 417A, 417C, 417D, 417F, 417G, 417H, 417I, 417K, 417M,417N, 417P, 417Q, 417S, 417W, 417Y, 418C, 418D, 418E, 418F, 418H, 418I,418K, 418N, 418Q, 418R, 418T, 418W, 418Y, 419C, 419D, 419E, 419F, 419G,419H, 419I, 419L, 419P, 419Q, 419S, 419T, 419Y, 420D, 420E, 420F, 420G,420H, 420I, 420K, 420L, 420M, 420N, 420Q, 420R, 420S, 420T, 420V, 420W,420Y, 421A, 421C, 421G, 421I, 421L, 421M, 421S, 421T, 422A, 422F, 422G,422H, 422I, 422M, 422N, 422Q, 422S, 422V, 422W, 422Y, 423A, 423D, 423G,423H, 423I, 423K, 423P, 423Q, 423R, 423T, 423V, 423W, 424D, 424E, 424G,424I, 424K, 424M, 424N, 424Q, 424R, 424S, 424T, 424V, 424W, 424Y, 425A,425I, 425K, 425L, 425M, 425S, 425T, 425V, 425W, and 425Y.

In some cases, the modification is a substitution of one or more aminoresidues present in the parental polypeptide to different amino acidresidues as exemplified by: 1A, 1D, 1F, 1G, 1H, 1K, 1M, 1N, 1Q, 1R, 1S,1T, 1V, 1W, 1Y, 2A, 2E, 2F, 2G, 2H, 2I, 2P, 2Q, 2R, 2S, 2W, 3D, 3E, 3F,3G, 3H, 3I, 3K, 3L, 3M, 3N, 3P, 3Q, 3R, 3S, 3V, 3W, 3Y, 4D, 4E, 4F, 4G,4I, 4K, 4L, 4Q, 4S, 4T, 4V, 4W, 5A, 5D, 5E, 5F, 5G, 5K, 5L, 5V, 5W, 6D,6H, 6K, 6L, 6P, 6Q, 6S, 6V, 6W, 7A, 7D, 7E, 7H, 7N, 7Q, 7R, 7S, 8A, 8C,8E, 8F, 8G, 8H, 8I, 8K, 8L, 8N, 8P, 8Q, 8R, 8T, 8V, 8W, 8Y, 9A, 9D, 9E,9F, 9H, 9I, 9K, 9M, 9N, 9P, 9R, 9V, 9W, 9Y, 10I, 10L, 10M, 10P, 10S,10y, 11A, 11F, 11M, 11V, 12I, 12M, 13A, 13D, 13Q, 14G, 14S, 14T, 14V,15F, 16M, 16Q, 18G, 18N, 18R, 19H, 19W, 20A, 20D, 20F, 20G, 20H, 20I,20K, 20M, 20R, 20S, 20V, 20W, 20Y, 21E, 21I, 21M, 21Q, 21S, 21V, 22I,22T, 22V, 23A, 23D, 23E, 23F, 23G, 23H, 23I, 23L, 23M, 23N, 23R, 23S,23T, 23V, 23W, 23Y, 24A, 24C, 24F, 24G, 24R, 24S, 24T, 24V, 24Y, 25E,25F, 25K, 25L, 25R, 25S, 25T, 25V, 25W, 25Y, 26I, 26L, 27A, 27E, 27F,27G, 27H, 27I, 27L, 27P, 27Q, 27R, 27S, 27T, 27V, 27W, 27Y, 28A, 28C,28G, 28H, 28I, 28K, 28L, 28M, 28N, 28P, 28Q, 28R, 28S, 28V, 28W, 28Y,29F, 29L, 29V, 30A, 30C, 30D, 30E, 30F, 30G, 30L, 30M, 30N, 30Q, 30R,30S, 30T, 30V, 30W, 30Y, 31A, 31F, 31G, 31H, 31I, 31K, 31L, 31M, 31N,31Q, 31S, 31T, 31V, 31Y, 32G, 32S, 33A, 33D, 33E, 33H, 33Q, 33S, 34W,35A, 35F, 35G, 35H, 35I, 35L, 35M, 35N, 35Q, 35R, 35S, 35V, 35W, 36F,36H, 36I, 36L, 36S, 36T, 36Y, 37L, 37V, 39A, 39P, 39S, 39V, 42V, 43A,43S, 43T, 43V, 44A, 44D, 44E, 44F, 44G, 44H, 44I, 44K, 44N, 44R, 44S,44T, 44Y, 45F, 45H, 45I, 45L, 45M, 45S, 45T, 46A, 46D, 46F, 46H, 46L,46M, 46N, 46R, 47A, 47D, 47F, 47G, 47H, 47I, 47K, 47L, 47N, 47P, 47R,47S, 47T, 47V, 47Y, 48A, 48E, 48F, 48H, 48N, 48P, 48W, 49A, 49F, 49G,49H, 49K, 49L, 49Q, 49R, 49S, 49T, 49V, 49W, 49Y, 50E, 50F, 50H, 50I,50K, 50L, 50M, 50P, 50R, 50S, 50T, 50V, 50W, 50Y, 51D, 51E, 51F, 51H,51I, 51K, 51L, 51P, 51Q, 51R, 51S, 51T, 51V, 51W, 52E, 52F, 52G, 52H,52I, 52K, 52L, 52M, 52N, 52Q, 52R, 52S, 52T, 52V, 52W, 52Y, 53A, 53E,53F, 53H, 53I, 53L, 53P, 53R, 53S, 53T, 53V, 54A, 54C, 54F, 54G, 54H,54L, 54N, 54P, 54R, 54T, 54W, 54Y, 55A, 55F, 55H, 55N, 55P, 55Q, 55S,55T, 55Y, 56D, 56E, 56F, 56G, 56I, 56K, 56L, 56P, 56Q, 56R, 56T, 56V,56W, 56Y, 57A, 57E, 57H, 57M, 57Q, 57R, 57S, 57Y, 58F, 59A, 59C, 59F,59H, 59N, 59P, 59R, 59S, 59T, 59W, 60L, 60N, 63H, 63N, 64A, 64S, 65A,65I, 65R, 66D, 66E, 66G, 66M, 66N, 66Q, 66R, 67A, 67F, 67G, 67I, 67L,67N, 67Q, 67T, 67W, 68D, 68F, 68H, 68I, 68L, 68N, 68R, 68S, 68T, 68V,68W, 69M, 69V, 72E, 72F, 72G, 72H, 72I, 72K, 72Q, 72S, 72T, 72V, 72W,72Y, 73F, 73M, 73W, 74M, 74T, 76A, 76L, 76M, 76P, 76Q, 76R, 76Y, 77A,77D, 77K, 77L, 77R, 77Y, 78D, 78E, 78F, 78G, 78H, 78I, 78K, 78L, 78P,78R, 78S, 78T, 78W, 78Y, 79A, 79M, 79Q, 79S, 80M, 81E, 81G, 81H, 81L,81M, 81N, 81Q, 81R, 81S, 81T, 81V, 81Y, 82D, 82F, 82G, 82I, 82K, 82L,82M, 82Q, 82R, 82S, 82T, 82Y, 83A, 83F, 83L, 84A, 84N, 84S, 84T, 85D,85E, 85F, 85G, 85I, 85K, 85R, 85S, 85T, 85V, 85W, 86D, 86E, 86F, 86G,86I, 86K, 86L, 86M, 86N, 86Q, 86R, 86S, 86V, 86W, 86Y, 87G, 88A, 88D,88F, 88G, 88H, 88K, 88L, 88M, 88N, 88Q, 88R, 88S, 88T, 88W, 88Y, 89D,89F, 89G, 89H, 89I, 89K, 89L, 89M, 89N, 89P, 89Q, 89R, 89S, 89T, 89V,89W, 89Y, 90D, 90E, 90F, 90H, 90I, 90K, 90M, 90N, 90R, 90S, 90T, 90V,90W, 91D, 91E, 91H, 91K, 91N, 91Q, 91R, 91S, 92L, 92V, 93A, 93D, 93G,93M, 93N, 93R, 93S, 93Y, 94I, 95F, 95M, 96I, 98C, 99I, 100C, 100F, 100M,100V, 103A, 103C, 103V, 104A, 104S, 105C, 105D, 105E, 105F, 105G, 105M,105W, 105Y, 106E, 106H, 106N, 106Q, 106S, 106T, 106Y, 107A, 107C, 107E,107F, 107G, 107H, 107I, 107K, 107L, 107M, 107N, 107P, 107Q, 107R, 107S,107T, 107V, 107W, 108C, 108D, 108E, 108F, 108G, 108H, 108I, 108K, 108L,108N, 108P, 108R, 108S, 108T, 108V, 108W, 108Y, 109D, 109H, 109I, 109K,109L, 109N, 109R, 109S, 109V, 109W, 109Y, 110V, 111C, 111E, 111F, 111G,111H, 111K, 111L, 111M, 111N, 111Q, 111R, 111T, 112A, 112D, 112E, 112H,112K, 112L, 112R, 112S, 112T, 112W, 112Y, 113A, 114L, 115A, 115H, 115I,115L, 115R, 115V, 115Y, 116A, 116F, 116G, 116H, 116I, 116L, 116N, 116Q,116R, 116T, 116V, 116W, 116Y, 118D, 118F, 118G, 118H, 118K, 118L, 118M,118N, 118Q, 118R, 118S, 118T, 118V, 118W, 118Y, 119E, 119F, 119I, 119K,119L, 119M, 119Q, 119S, 119T, 119Y, 121S, 124A, 124K, 124Q, 124R, 124S,124T, 125A, 125D, 125F, 125I, 125K, 125Q, 125R, 125V, 125Y, 126A, 126C,126D, 126F, 126G, 126H, 126I, 126K, 126L, 126N, 126P, 126R, 126S, 126T,126V, 126W, 126Y, 128A, 128C, 128E, 128F, 128G, 128H, 128I, 128L, 128M,128N, 128Q, 128R, 128S, 128T, 128V, 129C, 129D, 129E, 130A, 130F, 130L,130T, 130Y, 131A, 131C, 131D, 131F, 131G, 131H, 131I, 131K, 131L, 131N,131Q, 131T, 131V, 131W, 131Y, 132I, 132N, 132S, 132W, 134E, 134F, 134L,134M, 134R, 134Y, 135E, 136L, 140A, 141F, 141H, 142C, 142D, 142F, 142G,142H, 142I, 142K, 142M, 142Q, 142R, 142S, 142T, 142W, 142Y, 143C, 143D,143K, 143L, 143N, 143Q, 143S, 144T, 147F, 147L, 150H, 151C, 151D, 151E,151G, 151H, 151K, 151L, 151M, 151Q, 151S, 151T, 152A, 152C, 152E, 152F,152G, 152H, 152I, 152K, 152L, 152M, 152N, 152P, 152Q, 152R, 152S, 152V,152W, 152Y, 153E, 153F, 153H, 153K, 153L, 153N, 153R, 153T, 153V, 153W,153Y, 154A, 155M, 156A, 156F, 156G, 156K, 156L, 156Q, 156R, 156V, 156Y,157F, 157H, 158A, 158I, 158M, 158T, 158V, 159H, 159I, 159L, 159M, 160A,160C, 160D, 160E, 160F, 160G, 160H, 160I, 160K, 160L, 160M, 160Q, 160S,160T, 160V, 162I, 162M, 163A, 163E, 163F, 163G, 163H, 163I, 163K, 163L,163N, 163Q, 163R, 163S, 163T, 163V, 163W, 163Y, 164G, 164H, 164L, 164N,164S, 164T, 164V, 164W, 164Y, 165C, 165I, 165L, 165M, 165T, 165V, 166C,166I, 166M, 166V, 167A, 167C, 167E, 167F, 167G, 167I, 167K, 167L, 167M,167Q, 167R, 167S, 167T, 167V, 167W, 167Y, 168C, 168E, 168F, 168G, 168K,168L, 168M, 168N, 168S, 168T, 168V, 168W, 168Y, 170C, 171E, 171H, 171I,171M, 171N, 171Q, 171R, 172A, 175Y, 179A, 179C, 179G, 179H, 179S, 179W,180M, 181V, 184D, 186E, 187E, 187F, 187H, 187I, 187K, 187M, 187Q, 187S,187V, 187W, 188A, 188D, 188F, 188G, 188I, 188K, 188L, 188M, 188P, 188Q,188R, 188T, 188V, 189F, 189W, 190H, 190K, 190Q, 190R, 190S, 192G, 192K,192L, 192P, 192S, 192V, 195D, 195F, 195G, 195H, 195K, 195M, 195R, 195V,195W, 196A, 196C, 196E, 196F, 196H, 196I, 196K, 196L, 196M, 196Q, 196R,196S, 196T, 196V, 196Y, 197L, 197V, 198A, 198C, 198I, 198L, 198V, 199C,199D, 199E, 199F, 199H, 199R, 199S, 199T, 199Y, 200I, 200N, 200S, 200V,201C, 201D, 201E, 201F, 201G, 201H, 201I, 201K, 201L, 201N, 201Q, 201R,201T, 201V, 201W, 201Y, 202C, 202V, 203A, 203C, 203F, 203G, 203I, 203K,203L, 203Q, 203R, 203S, 203T, 203V, 203W, 203Y, 204I, 204M, 204W, 204Y,205A, 205C, 205I, 205L, 205M, 205N, 205V, 207A, 209L, 209V, 211H, 211S,211T, 212G, 212N, 213A, 213E, 213F, 213G, 213I, 213K, 213L, 213M, 213P,213Q, 213R, 213T, 213V, 214C, 214D, 214F, 214G, 214I, 214K, 214L, 214M,214N, 214Q, 214R, 214S, 214T, 214V, 214W, 214Y, 217I, 217Q, 217T, 218C,218D, 218E, 218F, 218G, 218H, 218I, 218K, 218L, 218M, 218P, 218Q, 218R,218S, 218T, 218V, 218W, 218Y, 219D, 219F, 219G, 219H, 219I, 219N, 219Q,219S, 219T, 219V, 219Y, 221C, 221E, 221G, 221Q, 221S, 221V, 222F, 222T,223H, 223L, 223M, 223W, 224I, 225E, 225F, 225N, 225P, 225Q, 225T, 225Y,226I, 226L, 229D, 229E, 229N, 229T, 230A, 230D, 230E, 230F, 230H, 230I,230K, 230M, 230Q, 230R, 230S, 230V, 230Y, 231H, 231W, 232S, 233A, 233D,233E, 233F, 233G, 233I, 233K, 233L, 233M, 233N, 233Q, 233S, 233T, 233V,233W, 233Y, 234A, 234F, 234G, 234H, 234I, 234L, 234M, 234N, 234Q, 234R,234T, 234V, 234W, 234Y, 235L, 235M, 236A, 236G, 236I, 236L, 236M, 236N,236Q, 237C, 237D, 237E, 237F, 237G, 237H, 237I, 237K, 237L, 237R, 237T,237V, 237W, 237Y, 238C, 238E, 238G, 238N, 238R, 238S, 238W, 239I, 239M,240A, 240E, 240F, 240G, 240L, 240Q, 240R, 240T, 240V, 240Y, 241F, 241G,241H, 241I, 241K, 241L, 241R, 241S, 241T, 241V, 241W, 241Y, 242A, 242C,242D, 242F, 242I, 242K, 242L, 242S, 242T, 242V, 242W, 242Y, 243D, 243E,243F, 243G, 243H, 243I, 243K, 243L, 243M, 243Q, 243R, 243S, 243T, 243V,243W, 243Y, 244I, 244M, 244V, 245C, 245F, 245H, 245I, 245L, 245M, 245N,245P, 245R, 245T, 245V, 245W, 245Y, 246C, 246D, 246E, 246G, 246I, 246L,246Q, 246W, 246Y, 247F, 247G, 247H, 247I, 247L, 247M, 247N, 247Q, 247T,247V, 247Y, 248F, 248G, 248K, 248L, 248Q, 248R, 248S, 248T, 248V, 248W,249A, 249C, 249F, 249L, 249M, 249V, 250C, 250E, 250F, 250G, 250H, 250I,250K, 250L, 250M, 250T, 250V, 250W, 250Y, 251A, 251C, 251D, 251E, 251G,251K, 251L, 251M, 251P, 251Q, 251V, 251Y, 252F, 252L, 252W, 253F, 253I,253K, 253L, 253M, 253R, 253T, 253W, 253Y, 254A, 254F, 254G, 254H, 254I,254L, 254N, 254T, 254V, 254Y, 255A, 255E, 255I, 255K, 255P, 255R, 255S,255V, 256A, 256C, 256I, 257E, 257I, 257L, 257P, 258C, 258D, 258E, 258N,258Q, 258R, 258S, 258V, 259A, 259G, 259H, 259K, 259Q, 259R, 259S, 259T,259W, 260A, 260C, 260D, 260F, 260H, 260N, 260Q, 260R, 260S, 260Y, 261M,262I, 263C, 263L, 263M, 263S, 263V, 264E, 264H, 264I, 264L, 264Y, 267A,267C, 267N, 267T, 268M, 268Q, 270F, 270G, 270N, 270S, 270V, 271F, 272G,272L, 272S, 272V, 273G, 273I, 273L, 273T, 273Y, 274F, 274G, 274H, 274I,274K, 274L, 274M, 274N, 274P, 274Q, 274R, 274S, 274T, 274V, 274W, 274Y,275F, 275G, 275H, 275K, 275P, 275Q, 275R, 275S, 275T, 275V, 276A, 276C,276D, 276F, 276G, 276H, 276I, 276K, 276L, 276M, 276N, 276P, 276Q, 276R,276S, 276T, 276Y, 277A, 277D, 277F, 277G, 277H, 277I, 277K, 277L, 277N,277P, 277Q, 277R, 277S, 277T, 277V, 277Y, 279H, 279K, 279L, 279M, 279N,279Q, 279Y, 280F, 280Y, 281C, 281L, 282A, 282D, 282I, 282K, 282L, 282M,282N, 282Q, 282T, 282W, 282Y, 283C, 283G, 283H, 283P, 283R, 283S, 283T,283V, 283W, 284A, 284C, 284E, 284F, 284G, 284H, 284I, 284K, 284L, 284N,284R, 284S, 284T, 284V, 284W, 284Y, 285E, 285M, 286C, 286L, 286M, 286V,287A, 287C, 287E, 287H, 287I, 287K, 287L, 287M, 287Q, 287S, 287T, 287V,288C, 288I, 288M, 288V, 289A, 290Y, 291C, 291G, 291L, 291S, 291T, 292A,292C, 292I, 292L, 292T, 293C, 293V, 294C, 294G, 294S, 294T, 295A, 295G,297D, 297E, 297F, 297G, 297H, 297I, 297K, 297L, 297M, 297N, 297P, 297Q,297R, 297T, 297V, 297W, 298C, 298D, 298E, 298F, 298H, 298I, 298K, 298L,298M, 298N, 298P, 298Q, 298R, 298S, 298V, 298W, 299C, 299G, 299I, 299N,299V, 300H, 300M, 300R, 300V, 301I, 301K, 301L, 301M, 301T, 302T, 303M,304L, 304Y, 305T, 305V, 307C, 307N, 308C, 308F, 308G, 308H, 308I, 308K,308L, 308M, 308N, 308P, 308Q, 308R, 308S, 308T, 308V, 308W, 308Y, 309D,309E, 309F, 309H, 309K, 309R, 309S, 310A, 311A, 311H, 311K, 311R, 312D,312F, 312G, 312H, 312I, 312K, 312L, 312M, 312P, 312Q, 312R, 312S, 312T,312V, 312W, 312Y, 313A, 313D, 313E, 313F, 313K, 313L, 313N, 313Q, 313R,313S, 313W, 313Y, 314A, 314F, 314H, 314K, 314L, 314M, 314Q, 314R, 314S,314T, 314W, 314Y, 315K, 315N, 315P, 315T, 316Y, 317A, 317C, 317E, 317F,317H, 317K, 317L, 317R, 317S, 317T, 317V, 317W, 317Y, 318D, 318F, 318H,318I, 318K, 318L, 318M, 318N, 318R, 318S, 318T, 318V, 318W, 318Y, 319G,319L, 319N, 319Q, 319V, 319W, 320C, 320F, 320G, 320I, 320K, 320L, 320M,320P, 320Q, 320T, 320V, 320Y, 321C, 321D, 321E, 321F, 321G, 321H, 321I,321K, 321L, 321R, 321S, 321T, 321V, 321W, 322L, 322M, 322V, 324A, 324F,324G, 324H, 324I, 324K, 324L, 324M, 324N, 324Q, 324R, 324S, 324T, 324V,324W, 324Y, 325C, 325D, 325G, 325H, 325I, 325K, 325L, 325M, 325N, 325P,325T, 325V, 327C, 327D, 327G, 327H, 327N, 327T, 328D, 328E, 328F, 328L,328N, 328Q, 328Y, 329F, 329H, 329Q, 330W, 330Y, 331D, 331F, 331G, 331I,331L, 331Q, 331S, 331T, 331V, 331Y, 332A, 332C, 332G, 332Q, 332S, 333C,333G, 333H, 333K, 333L, 333M, 333R, 333S, 333W, 333Y, 334D, 334H, 334I,334L, 334M, 334N, 334R, 334T, 335V, 336A, 336C, 336F, 336G, 336I, 336M,336N, 336Q, 336R, 336V, 336W, 336Y, 337H, 337N, 337S, 337V, 337W, 337Y,338G, 338I, 338L, 338M, 338S, 338T, 339C, 340F, 340H, 340K, 340L, 340M,340N, 340S, 340T, 340V, 340W, 341A, 341L, 341Y, 342A, 342K, 342N, 342R,342Y, 343A, 343D, 343E, 343F, 343H, 343K, 343L, 343M, 343Q, 343S, 343T,343W, 343Y, 344A, 344D, 344E, 344F, 344G, 344I, 344K, 344L, 344M, 344N,344Q, 344R, 344S, 344T, 344W, 344Y, 345C, 345E, 345F, 345G, 345H, 345I,345N, 345Q, 345S, 345T, 345V, 346C, 346D, 346E, 346I, 346K, 346L, 346M,346N, 346S, 346T, 346V, 346Y, 347D, 347F, 347H, 347I, 347K, 347L, 347M,347Q, 347R, 347S, 347T, 347V, 347W, 348F, 348H, 348I, 348K, 348R, 348S,348T, 348V, 348W, 348Y, 349A, 349F, 349G, 349I, 349K, 349M, 349N, 349R,349S, 349V, 349W, 349Y, 350D, 351A, 351D, 351G, 351H, 351K, 351L, 351M,351P, 351Q, 351R, 351T, 351V, 351W, 351Y, 352A, 352H, 352Q, 352T, 352Y,353A, 353D, 353E, 353G, 353I, 353K, 353L, 353M, 353Q, 353V, 353W, 353Y,355C, 355F, 355I, 355L, 355M, 355V, 355Y, 356D, 356F, 356G, 356I, 356K,356L, 356P, 356Q, 356T, 356W, 356Y, 357A, 357H, 357I, 357K, 357L, 357N,357Q, 357R, 357S, 357T, 357V, 357W, 357Y, 358C, 358D, 358F, 358G, 358H,358I, 358K, 358L, 358M, 358Q, 358R, 358S, 358T, 358V, 358Y, 359D, 359E,359H, 359L, 359M, 359P, 359Q, 359R, 359T, 359V, 359W, 360F, 360P, 360T,361C, 361L, 361M, 361N, 361Q, 361S, 361T, 361V, 362A, 362C, 362I, 362L,362V, 362Y, 363D, 363G, 363H, 363Q, 363R, 363S, 363V, 363W, 363Y, 364A,364C, 364G, 364I, 364L, 364M, 364Q, 364S, 364T, 364V, 365C, 365I, 365K,365L, 365N, 365R, 365S, 365V, 366A, 366K, 367L, 367M, 367N, 367R, 367S,367T, 367W, 367Y, 368G, 368I, 368K, 368L, 368R, 368T, 368V, 368W, 369C,369D, 369E, 369F, 369G, 369I, 369K, 369L, 369N, 369Q, 369S, 369T, 369V,369Y, 371A, 371C, 371F, 371I, 371L, 371M, 371N, 371S, 371T, 371Y, 372A,372C, 372I, 372L, 372N, 372S, 372T, 373A, 373C, 373F, 373I, 373M, 373T,373V, 374C, 374G, 374I, 374M, 374S, 374T, 374V, 375A, 375C, 375D, 375F,375H, 375L, 375M, 375Q, 375S, 375T, 375Y, 376G, 376I, 376S, 376T, 376V,377F, 377H, 377L, 377T, 377W, 377Y, 378C, 378E, 378F, 378G, 378H, 378I,378K, 378L, 378M, 378N, 378Q, 378R, 378T, 378V, 378W, 378Y, 379A, 379G,379H, 379I, 379K, 379L, 379Q, 379T, 379Y, 380C, 380E, 380F, 380G, 380H,380L, 380M, 380N, 380P, 380Q, 380R, 380T, 380V, 380W, 380Y, 381G, 381I,381Q, 381R, 381S, 381T, 381W, 381Y, 382A, 382C, 382F, 382I, 382K, 382Q,382R, 382T, 382W, 382Y, 383A, 383F, 383L, 383P, 383Q, 383V, 384A, 384G,384H, 384I, 384K, 384P, 384Q, 384V, 384W, 385C, 385F, 385H, 385I, 385K,385L, 385N, 385P, 385Q, 385R, 385S, 385V, 385W, 385Y, 386D, 386F, 386G,386H, 386L, 386N, 386R, 386S, 386T, 386V, 386W, 386Y, 387I, 387L, 388A,388C, 388G, 388H, 388L, 388P, 388S, 388T, 388W, 388Y, 389C, 389F, 389I,389M, 389Q, 389V, 390F, 390I, 390K, 390L, 390N, 390R, 390S, 390T, 390V,390W, 390Y, 391F, 391K, 391N, 391P, 391R, 391T, 391W, 391Y, 392A, 392C,392D, 392E, 392F, 392H, 392K, 392L, 392N, 392Q, 392R, 392S, 392V, 392Y,393A, 393C, 393D, 393F, 393G, 393H, 393I, 393L, 393Q, 393S, 393T, 393V,393W, 393Y, 394A, 394C, 394F, 394H, 394I, 394K, 394L, 394Q, 394V, 394W,395F, 395G, 395H, 395K, 395L, 395Q, 395R, 395S, 395T, 395V, 395W, 395Y,396C, 396D, 396S, 397C, 397D, 397F, 397G, 397H, 397I, 397L, 397P, 397S,397T, 397V, 397W, 398C, 398G, 398N, 398S, 398T, 398V, 399C, 399F, 399I,399K, 399L, 399R, 399S, 399T, 399V, 399W, 399Y, 400C, 400D, 400E, 400F,400G, 400H, 400I, 400K, 400L, 400M, 400Q, 400R, 400S, 400T, 400V, 400W,400Y, 401A, 401C, 401D, 401E, 401F, 401I, 401K, 401L, 401M, 401N, 401Q,401R, 401S, 401T, 401V, 401W, 401Y, 402A, 402C, 402D, 402E, 402F, 402G,402H, 402I, 402K, 402L, 402M, 402N, 402P, 402Q, 402R, 402T, 402V, 402W,402Y, 403A, 403C, 403H, 403I, 403M, 403V, 403W, 403Y, 404F, 404H, 404M,404R, 404T, 404V, 404W, 404Y, 405G, 405Q, 405S, 405T, 406L, 406T, 407F,407G, 407H, 407I, 407K, 407M, 407Q, 407R, 407S, 407T, 407V, 407W, 407Y,408D, 408E, 408F, 408N, 408V, 409C, 409F, 409I, 409L, 409R, 409T, 409V,409W, 409Y, 410V, 411E, 411F, 411M, 411Q, 411R, 411S, 411Y, 412N, 412T,413C, 413F, 413G, 413I, 413L, 413P, 413R, 413S, 413V, 413W, 413Y, 414H,414L, 414N, 414Q, 414T, 414V, 414W, 415D, 415E, 415G, 415I, 415R, 415V,415W, 416F, 416L, 416Q, 416Y, 417A, 417C, 417D, 417F, 417G, 417H, 417I,417K, 417M, 417N, 417Q, 418D, 418F, 418H, 418I, 418K, 418N, 418W, 418Y,419E, 419F, 419H, 419I, 419L, 419S, 419T, 420D, 420E, 420F, 420G, 420H,420I, 420K, 420L, 420Q, 420S, 420T, 420V, 420W, 420Y, 421C, 421L, 421M,421S, 421T, 422F, 422I, 422S, 422W, 423D, 423I, 423Q, 423R, 423T, 424M,424Q, 424R, 424V, 424Y, 425A, 425I, 425K, 425L, 425V, and 425Y.

In some case, the substitution are selected from 052D, 052E, 052I, 052K,052L, 052N, 052Q, 052R, 052V, 056D, 056E, 056I, 056K, 056L, 056N, 056Q,056R, 056V, 089D, 089E, 089I, 089K, 089L, 089N, 089Q, 089R, 089V, 152D,152E, 152I, 152K, 152L, 152N, 152Q, 152R, 152V, 153D, 153E, 153I, 153K,153L, 153N, 153Q, 153R, 153V, 201D, 201E, 201I, 201K, 201L, 201N, 201Q,201R, 201V, 251D, 251E, 251I, 251K, 251L, 251N, 251Q, 251R, 251V, 284D,284E, 284I, 284K, 284L, 284N, 284Q, 284R, 284V, 297D, 297E, 297I, 297K,297L, 297N, 297Q, 297R, 297V, 308D, 308E, 308I, 308K, 308L, 308N, 308Q,308R, 308V, 321D, 321E, 321I, 321K, 321L, 321N, 321Q, 321R, 321V, 328D,328E, 328I, 328K, 328L, 328N, 328Q, 328R, 328V, 347D, 347E, 347I, 347K,347L, 347N, 347Q, 347R, 347V, 357D, 357E, 357I, 357K, 357L, 357N, 357Q,357R, 357V, 359D, 359E, 359I, 359K, 359L, 359N, 359Q, 359R, 359V, 369D,369E, 369I, 369K, 369L, 369N, 369Q, 369R, 369V, 385D, 385E, 385I, 385K,385L, 385N, 385Q, 385R, 385V, 388D, 388E, 388I, 388K, 388L, 388N, 388Q,388R, 388V, 391D, 391E, 391I, 391K, 391L, 391N, 391Q, 391R, 391V, 400D,400E, 400I, 400K, 400L, 400N, 400Q, 400R, 400V, 416D, 416E, 416I, 416K,416L, 416N, 416Q, 416R, and 416V, which mutations have PI values >0.5for both protein and activity.

Substitutions that changed the amino acid residue present at position153 of the parental AmyE polypeptide to N, K or F exhibited increasedability to convert maltose and maltoheptaose substrates to glucose.Substitutions that changed the amino acid residue present at position153 of the parental AmyE polypeptide to K exhibited increased ability toconvert a DP7 substrate to glucose.

The substitutions L142F, L142G, L142Q, L142S, L142W, L142Y, A2141,A214V, S245Y, Q126F, Q126L, Q126P, Q126V, S131L, and S2541, improvedstarch liquefaction performance in the context of the full-length AmyEpolypeptide. The substitutions W60L, W60M, W60N, I100F, I100M, S105M,S105W, G207A, T270A, T270E, T270L, T270N, T270V, and T279A, improvedstarch liquefaction performance in the context of the truncated AmyEpolypeptide.

Substitutions as one or more of positions 052D, 052E, 052I, 052K, 052L,052N, 052Q, 052R, 052V, 056D, 056E, 056I, 056K, 056L, 056N, 056Q, 056R,056V, 089D, 089E, 089I, 089K, 089L, 089N, 089Q, 089R, 089V, 152D, 152E,152I, 152K, 152L, 152N, 152Q, 152R, 152V, 153D, 153E, 153I, 153K, 153L,153N, 153Q, 153R, 153V, 201D, 201E, 201I, 201K, 201L, 201N, 201Q, 201R,201V, 251D, 251E, 251I, 251K, 251L, 251N, 251Q, 251R, 251V, 284D, 284E,284I, 284K, 284L, 284N, 284Q, 284R, 284V, 297D, 297E, 297I, 297K, 297L,297N, 297Q, 297R, 297V, 308D, 308E, 308I, 308K, 308L, 308N, 308Q, 308R,308V, 321D, 321E, 321I, 321K, 321L, 321N, 321Q, 321R, 321V, 328D, 328E,328I, 328K, 328L, 328N, 328Q, 328R, 328V, 347D, 347E, 347I, 347K, 347L,347N, 347Q, 347R, 347V, 357D, 357E, 357I, 357K, 357L, 357N, 357Q, 357R,357V, 359D, 359E, 359I, 359K, 359L, 359N, 359Q, 359R, 359V, 369D, 369E,369I, 369K, 369L, 369N, 369Q, 369R, 369V, 385D, 385E, 385I, 385K, 385L,385N, 385Q, 385R, 385V, 388D, 388E, 388I, 388K, 388L, 388N, 388Q, 388R,388V, 391D, 391E, 391I, 391K, 391L, 391N, 391Q, 391R, 391V, 400D, 400E,400I, 400K, 400L, 400N, 400Q, 400R, 400V, 416D, 416E, 416I, 416K, 416L,416N, 416Q, 416R, and 416V, have PI values >0.5 for both protein andactivity, and are expected to be combinable for affecting variousproperties of AmyE polypeptides.

While many position of the AmyE polypeptides can be mutated, positions75, 97, 101, 102, 120, 123, 133, 137, 182, 266, and 306, of AmyEpolypeptides were restrictive, in the sense that mutations at theseposition generally decrease performance. In particular, positions 75 and123 were determined to be fully restrictive for performance in atruncated version of the parental polypeptide, while positions 75, 97,101, 102, 120, 133, 137, 182, 266, and 306 were determined to be fullyrestrictive for performance in a full-length version of the parentalpolypeptide.

Note that while many mutation are listed in the context of large groupsand subgroups, each mutation is a separate entity and any one or more ofthe identified mutations can be included or excluded from a furthersubgroup of mutations. According, the composition and methods includeAmyE variants having any one or more variants described herein, orcombinations thereof.

3. Production of AmyE Polypeptides

A DNA sequence encoding an AmyE polypeptide can be expressed, in enzymeform, using an expression vector which typically includes controlsequences encoding a suitable promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a repressor gene orvarious activator genes.

Vectors comprising the nucleic acids encoding AmyE or variants thereofalso are provided. Host cells comprising the vectors are provided. Thehost cell may express the polynucleotide encoding the AmyE variant. Thehost may be a Bacillus sp., e.g., B. subtilis.

3.1 Polynucleotides and Vectors

Aspect of the present compositions and methods include polynucleotidesencoding AmyE polypeptides, as well as vectors and host cells useful forexpressing AmyE polypeptides based on such polynucleotides. Nucleicacids encoding AmyE polypeptides include, but are not limited to, thepolynucleotides of SEQ ID NO: 5 and SEQ ID NO: 6, which encode the AmyEof SEQ ID NO: 1 and AmyE-tr (SEQ ID NO: 2), respectively, and variantsthereof. Further representative polynucleotides include that of SEQ IDNO: 7, which encodes Amy31A (SEQ ID NO: 3). The AmyE disclosed in NCBIAccession Nos. ABK54355, AAF14358, AAT01440, AAZ30064, NP_(—)388186,AAQ83841, and BAA31528 are similarly encoded by polynucleotidesdisclosed in publicly accessible databases, which sequences areincorporated herein by reference. Nucleic acids may be DNA, mRNA, orcDNA sequences. Nucleic acids further include degenerate sequencescorresponding to any of the aforementioned nucleic acids. Degeneratesequences may be designed for optimal expression by using codonspreferred by a particular host organism.

Recombinant expression vector carrying DNA sequence encoding AmyEpolypeptides (including variants) may be any vector that mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e., a vector that exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, mini-chromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated. The integrated gene may also be amplified to createmultiple copies of the gene in the chromosome by use of an amplifiableconstruct driven by antibiotic selection or other selective pressure,such as an essential regulatory gene or by complementation of anessential metabolic pathway gene.

An expression vector typically includes the components of a cloningvector, e.g., an element that permits autonomous replication of thevector in the selected host organism and one or more phenotypicallydetectable markers for selection purposes. The expression vectornormally comprises control nucleotide sequences encoding a promoter,operator, ribosome binding site, translation initiation signal andoptionally, a repressor gene or one or more activator genes. In oneaspect, all the signal sequences used target the material to the cellculture media for easier enzyme collection and optionally purification.The procedures used to ligate the DNA construct encoding an AmyE orvariant thereof, the promoter, terminator and other elements,respectively, and to insert them into suitable vectors containing theinformation necessary for replication, are well known to persons skilledin the art (see e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2^(nd) ed., Cold Spring Harbor, 1989 and 3^(rd) ed., 2001).

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence thatshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Suitable promoters for directing the transcription ofthe DNA sequence encoding an AmyE or variant thereof, especially in abacterial host, include various Bacillus-derived promoters, such as anα-amylase promoter derived from B. subtilis, B. licheniformis, B.stearothermophilus, or B. amyloliquefaciens, the promoter of the lacoperon of E. coli, the Streptomyces coelicolor agarase gene dagA or celApromoters, and the promoters of the Bacillus subtilis xylA and xylBgenes, etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase, or A. nidulans acetamidase. When thegene encoding the AmyE or variant thereof is expressed in a bacterialspecies such as E. coli, a suitable promoter can be selected, forexample, from a bacteriophage promoter including a T7 promoter and aphage lambda promoter. Examples of suitable promoters for the expressionin a yeast species include but are not limited to the Gal 1 and Gal 10promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 orAOX2 promoters.

The expression vector may also comprise a suitable transcriptionterminator and, in eukaryotes, polyadenylation sequences operablyconnected to the DNA sequence encoding the α-amylase variant.Termination and polyadenylation sequences may suitably be derived fromthe same sources as the promoter. The vector may further comprise a DNAsequence enabling the vector to replicate in the host cell in question.Examples of such sequences are the origins of replication of plasmidspUC19, pACYC177, pUB110, pE194, pAMB1, pICatH, and pIJ702.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or a gene which confersantibiotic resistance, e.g., ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD, and xxsC, a marker givingrise to hygromycin resistance, or the selection may be accomplished byco-transformation as known in the art. See, e.g., WO 91/17243.

3.2 AmyE Polypeptide Expression and Host Organisms

It is generally advantageous if the AmyE polypeptide is secreted intothe culture medium, when expressed in a host cell. To this end, the AmyEpolypeptide may comprise a signal sequence that permits secretion of theexpressed enzyme into the culture medium. The signal sequence mayencoded by the same gene as the AmyE. For example, the AmyE set forth inSEQ ID NO: 1 is expressed naturally with a signal sequence andadditional N-terminal amino acids having the sequenceMFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASAETANKSNE (SEQ ID NO: 9). The signalsequence alternatively may be a B. subtilis sp. signal sequence from adifferent AmyE or even a different protein. Further, the signal sequencemay be from a different species, e.g., B. licheniformis. The signalsequence may be chosen to provide optimal expression of the AmyE orvariant thereof in a particular host cell, for example. The mature AmyEmay be produced as a result of proteolytic cleavage of additionalsequences from the N-terminus that are not signal sequences. Forexample, a 31-amino acid residue signal sequence from B. licheniformis(“LAT leader sequence”) may be fused in frame with an AmyE sequence. Forexample, a nucleic acid encoding AmyE is operably linked to a B.licheniformis signal sequence in the expression vector shown in FIG. 2.

An isolated cell, either comprising a DNA construct or an expressionvector, is advantageously used as a host cell in the recombinantproduction of an AmyE or variant thereof. The cell may be transformedwith the DNA construct encoding the AmyE or variant thereof, optionallyby integrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

Examples of suitable bacterial host organisms are Gram positivebacterial species such as Bacillaceae, including B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. lautus, B.megaterium, and B. thuringiensis; Streptomyces spp., such as S. murinus;lactic acid bacterial species including Lactococcus spp., such as L.lactis; Lactobacillus spp., including L. reuteri; Leuconostoc spp.;Pediococcus spp.; and Streptococcus spp. Still other useful hostsinclude Bacillus spp. A 7-7, for example. Alternatively, strains of aGram negative bacterial species belonging to Enterobacteriaceae,including E. coli, or to Pseudomonadaceae can be selected as the hostorganism.

A suitable yeast host organism can be selected from biotechnologicallyrelevant yeasts species, such as, but not limited to, Pichia spp.,Hansenula spp., Kluyveromyces spp., Yarrowinia spp., Saccharomyces spp.,including S. cerevisiae, or a species belonging to Schizosaccharomyces,such as S. pombe. A strain of the methylotrophic yeast species Pichiapastoris can be used as the host organism. Alternatively, the hostorganism can be a Hansenula spp. Suitable host organisms amongfilamentous fungi include species of Aspergillus, e.g., A. niger, A.oryzae, A. tubigensis, A. awamori, or A. nidulans. Alternatively, astrain of Fusarium spp., e.g., Fusarium oxysporum or Rhizomucor spp.,such as R. miehei, can be used as the host organism. Other suitableyeasts include Thermomyces spp. and Mucor spp. Fungal cells may betransformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known in the art. A suitable procedure for transformingAspergillus host cells, for example, is described in EP 238023.

An aspect of the present compositions and methods is a method ofproducing an AmyE variant, which method comprises cultivating a hostcell as described above under conditions conducive to the production ofthe variant and recovering the variant from the cells and/or culturemedium. The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the AmyE variant. Suitable media and media components areavailable from commercial suppliers or may be prepared according topublished recipes, e.g., as described in catalogues of the American TypeCulture Collection (ATCC). Exemplary culture media include, but are notlimited to, those for fed-batch fermentations performed in a threethousand liter (3,000 L) stirred tank fermentor. The growth medium inthat case can consist of corn steep solids and soy flour as sources oforganic compounds, along with inorganic salts as a source of sodium,potassium, phosphate, magnesium and sulfate, as well as trace elements.Typically, a carbohydrate source such as glucose is also part of theinitial medium. Once the culture has established itself and beginsgrowing, the carbohydrate is metered into the tank to maintain theculture as is known in the art. Samples are removed from the fermentorat regular intervals to measure enzyme titer using, for example, acolorimetric assay method. The fermentation process is halted when theenzyme production rate stops increasing according to the measurements.

An AmyE polypeptides secreted from the host cells may conveniently berecovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulfate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Host cells may be cultured under suitable conditions that allowexpression of the AmyE polypeptides. Expression of the proteins may beconstitutive such that they are continually produced, or inducible,requiring a stimulus to initiate expression. In the case of inducibleexpression, protein production can be initiated when required byaddition of an inducer substance, e.g., dexamethasone, IPTG, orSepharose, to the culture medium, for example. Polypeptides can also beproduced recombinantly in an in vitro cell-free system, such as the TNT™(Promega) rabbit reticulocyte system.

A host for expressing an AmyE polypeptides can be cultured under aerobicconditions in the appropriate medium for the host. Shaking or acombination of agitation and aeration can be provided, with productionoccurring at the appropriate temperature for that host, e.g., from about30° C. to about 75° C., depending on the needs of the host andproduction of the desired α-amylase variant. Culturing can occur fromabout 12 to about 100 hours or greater (and any hour value therebetween) or more particularly from 24 to 72 hours. Typically, theculture broth is at a pH of about 5.5 to about 8.0, again depending onthe culture conditions needed for the host cell relative to productionof the AmyE variant.

The amylolytic activity of the expressed enzyme may be determined using,e.g., potato starch as substrate. This method is based on the break-downof modified potato starch by the enzyme, and the reaction is followed bymixing samples of the starch/enzyme solution with an iodine solution.Initially, a blackish-blue color is formed, but during the break-down ofthe starch the blue color gets weaker and gradually turns into areddish-brown, which is compared to a colored glass standard.

AmyE polypeptides may be expressed as a fusion protein that comprisessequences at the N- and/or C-terminus of the mature form of AmyE thatfacilitate expression, detection, and/or purification, e.g., a signalsequence or a His-tag. Such a sequence includes a signal sequence, whichfacilitates secretion and expression of the AmyE in a host organism.Additional amino acid residues may be cleaved from the N-terminus of anAmyE, following cleavage of the signal sequence, as discussed in Yang etal., “Nucleotide sequence of the amylase gene from Bacillus subtilis,”Nucleic Acids Res. 11: 237-49 (1983).

4. Purification of AmyE Polypeptides

In some cases, conventional methods can be used in order to prepare apurified AmyE polypeptides. After growing a host organism in culture, agrowth (or “fermentation”) broth is obtained, and the microbial cellsand various suspended solids, including residual raw fermentationmaterials, are removed by conventional separation techniques to obtainan amylase solution. Filtration, centrifugation, microfiltration, rotaryvacuum drum filtration, followed by ultra-filtration, extraction orchromatography, or the like are generally used.

It is generally desirable to concentrate the solution containing theexpressed AmyE or variant thereof to optimize recovery, since the use ofun-concentrated solutions requires increased incubation time to collectprecipitates containing the purified enzyme. The solution isconcentrated using conventional techniques until the desired enzymelevel is obtained. Concentration of the enzyme containing solution maybe achieved by any of the techniques discussed above. In one embodiment,rotary vacuum evaporation and/or ultrafiltration is used. Alternatively,ultrafiltration can be used.

Precipitation can be performed using, for example, a metal halideprecipitation agent. Metal halide precipitation agents include: alkalimetal chlorides, alkali metal bromides and blends of two or more ofthese metal halides. The metal halide may be selected from the groupconsisting of sodium chloride, potassium chloride, sodium bromide,potassium bromide and blends of two or more of these metal halides.Suitable metal halides include sodium chloride and potassium chloride,particularly sodium chloride, which can further be used as apreservative. The selection of conditions of the precipitation formaximum recovery, including incubation time, pH, temperature andconcentration of AmyE or variant thereof, will be readily apparent toone of ordinary skill in the art after routine testing.

Generally, at least about 5% w/v (weight/volume) to about 25% w/v ofmetal halide is added to the concentrated enzyme variant solution, andusually at least 8% w/v. Generally, no more than about 25% w/v of metalhalide is added to the concentrated enzyme variant solution and usuallyno more than about 20% w/v. The optimal concentration of the metalhalide precipitation agent will depend, among others, on the nature ofthe specific AmyE or variant thereof and on its concentration insolution.

An alternative to effect precipitation of the enzyme is to use oforganic compounds, which can be added to the concentrated enzyme variantsolution. The organic compound precipitating agent can include:4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid,alkyl esters of 4-hydroxybenzoic acid, and blends of two or more ofthese organic compounds. The addition of said organic compoundprecipitation agents can take place prior to, simultaneously with orsubsequent to the addition of the metal halide precipitation agent, andthe addition of both precipitation agents, organic compound and metalhalide, may be carried out sequentially or simultaneously. For furtherdescriptions, see, e.g., U.S. Pat. No. 5,281,526 to Danisco A/S, forexample.

Generally, the organic compound precipitation agents are selected fromthe group consisting of alkali metal salts of 4-hydroxybenzoic acid,such as sodium or potassium salts, and linear or branched alkyl estersof 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12carbon atoms, and blends of two or more of these organic compounds. Theorganic compound precipitations agents can be for example linear orbranched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl groupcontains from 1 to 10 carbon atoms, and blends of two or more of theseorganic compounds. Suitable organic compounds include linear alkylesters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1to 6 carbon atoms, and blends of two or more of these organic compounds.Methyl esters of 4-hydroxybenzoic acid, propyl ester of 4-hydroxybenzoicacid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of4-hydroxybenzoic acid and blends of two or more of these organiccompounds can also be used. Additional organic compounds also include,but are not limited to, 4-hydroxybenzoic acid methyl ester (methylPARABEN) and 4-hydroxybenzoic acid propyl ester (propyl PARABEN), whichare also amylase preservative agents. Addition of the such an organiccompound precipitation agent provides the advantage of high flexibilityof the precipitation conditions with respect to pH, temperature, enzymeconcentration, precipitation agent concentration, and time ofincubation. Generally, at least 0.01% w/v of organic compoundprecipitation agent is added to the concentrated enzyme variant solutionand usually at least 0.02% w/v. Generally, no more than 0.3% w/v oforganic compound precipitation agent is added to the concentrated enzymevariant solution and usually no more than 0.2% w/v.

The concentrated enzyme solution, containing the metal halideprecipitation agent and, in one aspect, the organic compoundprecipitation agent, is adjusted to a pH that necessarily will depend onthe enzyme variant to be purified. Generally, the pH is adjusted to alevel near the isoelectric point (pI) of the amylase. For example, thepH can be adjusted within a range of about 2.5 pH units below the pI toabout 2.5 pH units above the pI. The pH may be adjusted accordingly ifthe pI of the variant differs from the wild-type pI.

The incubation time necessary to obtain a purified enzyme precipitatedepends on the nature of the specific enzyme, the concentration ofenzyme, and the specific precipitation agent(s) and its (their)concentration. Generally, the time effective to precipitate the enzymevariant is between about 1 to about 30 hours; usually it does not exceedabout 25 hours. In the presence of the organic compound precipitationagent, the time of incubation can still be reduced to less than about 10hours, and in most cases even about 6 hours.

Generally, the temperature during incubation is between about 4° C. andabout 50° C. Usually, the method is carried out at a temperature betweenabout 10° C. and about 45° C., and particularly between about 20° C. andabout 40° C. The optimal temperature for inducing precipitation variesaccording to the solution conditions and the enzyme or precipitationagent(s) used.

The overall recovery of purified enzyme precipitate, and the efficiencywith which the process is conducted, is improved by agitating thesolution comprising the enzyme, the added metal halide and the addedorganic compound. The agitation step is done both during addition of themetal halide and the organic compound, and during the subsequentincubation period. Suitable agitation methods include mechanicalstiffing or shaking, vigorous aeration, or any similar technique.

The purified enzyme may be further purified by conventional separationtechniques, such as filtration, centrifugation, microfiltration, rotaryvacuum filtration, ultrafiltration, press filtration, cross membranemicrofiltration, cross flow membrane microfiltration, or the like. Crossmembrane microfiltration can be one method used. Further purification ofthe purified enzyme precipitate can be obtained by washing theprecipitate with water. For example, the purified enzyme precipitate maybe washed with water containing the metal halide precipitation agent,for example, with water containing the metal halide and the organiccompound precipitation agents.

During culturing, expressed enzyme may accumulate in the culture broth.For the isolation and purification of the expressed enzyme, the culturebroth may be centrifuged or filtered to eliminate cells, and theresulting cell-free liquid may be used for the purification of theenzyme. In one embodiment, the cell-free broth is subjected to saltingout using ammonium sulfate at about 70% saturation; the 70%saturation-precipitation fraction is then dissolved in a buffer andapplied to a column such as a Sephadex G-100 column, and eluted torecover the enzyme active fraction. For further purification, aconventional procedure such as ion exchange chromatography may be used.

Purified enzymes are useful for all applications in which the enzyme aregenerally utilized. For example, they can be used in laundry detergentsand spot removers, in the food industry, in starch processing andbaking, and in pharmaceutical compositions as digestive aids. They canbe made into a final product that is either liquid (solution, slurry) orsolid (granular, powder).

Alternatively, the enzyme product can be recovered and a flocculatingagent is added to the media in order to remove cells and cell debris byfiltration or centrifugation without further purification of the enzyme.

AmyE polypeptides produced and purified by the methods described abovecan be used in a variety of useful industrial applications. The enzymespossess valuable properties facilitating applications related to fabricand household care (F&HC). For example, AmyE polypeptides can be used asa component in washing, dishwashing and hard-surface cleaning detergentcompositions. AmyE polypeptides are also useful in the production ofsweeteners and ethanol from starch, and/or for textile desizing. AmyEpolypeptides are particularly useful in starch-conversion processes,including starch liquefaction and/or saccharification processes, asdescribed, for example, in WO 2005/111203 and U.S. Published ApplicationNo. 2006/0014265 (Danisco A/S). These uses of AmyE polypeptides aredescribed in more detail below.

5. Compositions and Methods of use of AmyE Polypeptides

5.1. Starch Processing Compositions and Methods

5.1.1. Overview

AmyE polypeptides can be utilized for starch processing/conversion,which is central to producing sweeteners, producing alcohol for fuel ordrinking (i.e., potable alcohol), producing a beverage, processing canesugar, or producing desired organic compounds, e.g., citric acid,itaconic acid, lactic acid, gluconic acid, ketones, amino acids,antibiotics, enzymes, vitamins, hormones, and the like. Starchconversion generally involves the hydrolysis of a slurry of gelatinizedor granular starch into a soluble starch hydrolysate. Conventions starchconversion involves three consecutive enzymatic processes: aliquefaction process, a saccharification process, and a further processto produce a desired product from glucose. Depending on the desiredproduct, the further process may be isomerization, fermentation, and thelike. In the process of converting starch to fructose syrup, the furtherprocess is isomerization.

5.1.2. Starch Compositions

The starch to be processed may be obtained from tubers, roots, stems,legumes, cereals or whole grain. More specifically, the granular starchmay be obtained from corns, cobs, wheat, barley, rye, milo, sago,cassava, tapioca, sorghum, rice, peas, bean, banana, or potatoes.Specially contemplated are both waxy and non-waxy types of corn andbarley. The starch may be a highly refined starch quality, for instance,at least 90%, at least 95%, at least 97%, or at least 99.5% pure.Alternatively, the starch can be a more crude starch containing materialcomprising milled whole grain, including non-starch fractions such asgerm residues and fibers. The raw material, such as whole grain, ismilled to open up the structure and allow further processing.

Two milling processes are suitable: wet and dry milling (dry grinding).In dry milling, the whole kernel is milled and used. Dry milled grainmay include significant amounts of non-starch carbohydrate compounds, inaddition to starch. When such a heterogeneous material is processed byjet cooking, often only a partial gelatinization of the starch isachieved. Wet milling gives a good separation of germ and meal (starchgranules and protein) and is usually used in the production of syrups.The process may be conducted in an ultrafiltration system where theretentate is held under recirculation in presence of enzymes, raw starchand water, where the permeate is the soluble starch hydrolysate. Theprocess may also be conducted in a continuous membrane reactor withultrafiltration membranes, where the retentate is held underrecirculation in presence of enzymes, raw starch and water, and wherethe permeate is the soluble starch hydrolysate. The process may furtherbe conducted in a continuous membrane reactor with microfiltrationmembranes and where the retentate is held under recirculation inpresence of enzymes, raw starch and water, and where the permeate is thesoluble starch hydrolysate.

The starch slurry to be used in any of the above aspects may have about20% to about 55% dry solids granular starch, about 25% to about 40% drysolids granular starch, or about 30% to about 35% dry solids granularstarch. The enzyme variant converts the soluble starch into a solublestarch hydrolysate of the granular starch in the amount of at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%.

5.1.3. Liquefaction and Saccharification

During the liquefaction step, long-chained starch molecules present inthe starch slurry are degraded into shorter branched and linearmolecules (maltodextrins) by an α-amylase. Numerous α-amylases areavailable commercially, including SPEZYME® Xtra (Genencor) andLIQUOZYME® (Novozymes).

The liquefaction process is generally carried out at about 105-110° C.for about 5 to 10 minutes followed by 1-2 hours at 95° C. The pH ofliquefaction is typically between about 5.0 and about 6.2, and usuallyabove 5.5. To promote α-amylase stability under these conditions, 1 mMof calcium is typically added (40 ppm free calcium ions). Following thistreatment, the liquefied starch composition will largely containdextrins, and will have a “dextrose equivalent” (DE) of about 10-15.

Following the liquefaction process, the dextrins are typicallyconverted, in a separate saccharification step, into dextrose, byaddition of a glucoamylase (e.g., AMG™). A debranching enzyme, such asan isoamylase or a pullulanase (e.g., PROMOZYME®) may also be added. Toprepare for the saccharification step, the pH of the slurry is typicallyreduced to a value below about 4.5, while maintaining the temperature at95° C. or more, such that the liquefying α-amylase is denatured. Thetemperature then is lowered to about 60° C., and the glucoamylase and adebranching enzyme are added to affect glucose production from dextrins.The saccharification process proceeds typically for about 24 to about 72hours.

5.1.4. Further Processing of Glucose Produced by Saccharification

After the saccharification process, the dextrose syrup may be convertedinto high fructose syrup using an immobilized glucose isomerase (such asSWEETZYME®), for example. In one regard, the soluble starch hydrolysateof the process is subjected to conversion into high fructosestarch-based syrup (HFSS), such as high fructose corn syrup (HFCS). Thisconversion can be achieved using a glucose isomerase, particularly aglucose isomerase immobilized on a solid support. Contemplatedisomerases included the commercial products SWEETZYME®, IT (NovozymesA/S); G-ZYME® IMGI, and G-ZYME® G993, KETOMAX®, G-ZYME® G993, G-ZYME®G993 liquid, and GENSWEET® IGI. While Ca²⁺ increases the stability ofconventional α-amylases, it strongly inhibits the activity of theglucose isomerase. Thus, Ca²⁺ is typically removed prior toisomerization, e.g., by ion exchange, such that the level of Ca²⁺ isbelow 3-5 ppm. This process is time consuming and expensive.

Alternatively, glucose produced by saccharification is used forfermentation to produce a fermentation product, e.g., ethanol, butanol,and other compound described herein and known in the art. A typicalcomplete process for producing ethanol from starch-containing materialby fermentation comprises: (i) liquefying the starch-containing materialwith an AmyE or variant thereof; (ii) saccharifying the liquefied mashobtained; and (iii) fermenting the material obtained in step (ii) in thepresence of a fermenting organism. Optionally the process furthercomprises recovery of the ethanol. During the fermentation, the ethanolcontent reaches at least about 7%, at least about 8%, at least about 9%,at least about 10% such as at least about 11%, at least about 12%, atleast about 13%, at least about 14%, at least 15%, or at least 16%ethanol.

The saccharification and fermentation processes may be carried out as asimultaneous saccharification and fermentation (SSF) process. Whenfermentation is performed simultaneously with the hydrolysis, thetemperature can be between 30° C. and 35° C., particularly between 31°C. and 34° C. The process may be conducted in an ultrafiltration systemwhere the retentate is held under recirculation in presence of enzymes,raw starch, yeast, yeast nutrients and water and where the permeate isan ethanol containing liquid. Also contemplated is the process conductedin a continuous membrane reactor with ultrafiltration membranes andwhere the retentate is held under recirculation in presence of enzymes,raw starch, yeast, yeast nutrients and water and where the permeate isan ethanol containing liquid.

Glucose produced by saccharification may also be used for production ofa fermentation product such as ethanol, butanol, citric acid, monosodiumglutamate, gluconic acid, sodium gluconate, calcium gluconate, potassiumgluconate, glucono delta-lactone, or sodium erythorbate.

5.1.5. Advantages of AmyE α-Amylase

AmyE polypeptides offer several advantages when used in starchhydrolysis, which distinguish them from other α-amylases and allow forthe streamlining of starch hydrolysis methods. First, dextrins may beconverted into dextrose by AmyE polypeptides under the same reactionconditions that are suitable for glucoamylases. This obviates the needto optimize the reaction mixture (e.g., slurry) pH and temperature foran α-amylase, and then adjust the reactions conditions to accommodate aglucoamylase, e.g., by reducing the pH and/or temperature of the slurry.In this manner, the use of AmyE in starch hydrolysis permitsliquefaction and saccharification to be performed under the same slurryconditions, thereby eliminating a step in starch hydrolysis.

In some cases, both liquefaction and saccharification can be performedat a single pH between about 4-7, e.g., about 4-5, about 4-6, about 5-6,about 5-7, and about 6-7. Note that the single pH criterion ignores anyminor changes to the pH of the reaction mixture that occur duringliquefaction or saccharification, but which do not involve to theaddition of acid or base to intentionally change the pH of the reactionmixture. In some cases, both liquefaction and saccharification can beperformed at a pH below that which conventional liquefaction processesare performed, for example, a pH of less than about 5.0, less than about4.8, less than about 4.6, less than about 4.4, less than about 4.2, oreven a pH of about 4.0. The use of AmyE for liquefaction also allows theinclusion of a higher percentage of thin stillage in the starchcomposition used for liquefaction, for example, >50%, or even >60% ofthe reaction mix. In some cases, both liquefaction and saccharificationcan be performed at a temperature of about 20-105° C., for example,60-85° C., or a temperature that is about 10, 12, 14, 16, 18, or even20° C. below the starch gelation temperature (i.e., about 75° C.).Importantly, liquefaction and saccharification can be performed entirelywithout an intervening pH adjustment. Alternatively, where pH adjustmentis still desirable between liquefaction and saccharification, it can beperformed using a reduced amount of acid or base, compared to the amountused in a conventional process, thereby introducing less salt in to thereaction mix.

In addition, AmyE polypeptides catalyze the breakdown of complex sugars,such as maltose, maltotriose, and maltoheptaose, into glucose. Such anenzymatic activity is conventionally associated with glucoamylasesrather than α-amylases. This activity of AmyE polypeptides allows starchhydrolysis to glucose to be performed either in the absence of aseparate glucoamylase, or in the presence of a reduced amount of aglucoamylase, compared to the amount required using a conventionalα-amylase. In this manner, the use of AmyE polypeptides in starchhydrolysis permits liquefaction and saccharification to be performedsimultaneously using a single enzyme that functions as both an α-amylaseand a glucoamylase, thereby eliminating or reducing the need forseparate enzymes.

AmyE polypeptides also require little or no Ca²⁺ for stability, reducingor eliminating the need to add Ca²⁺ to a liquefaction reaction. Inaddition to avoiding the step of adding Ca²⁺, this avoids the need tosubsequently remove Ca²⁺ from a slurry (e.g., by ion exchange) prior tocontacting the slurry with an enzyme such as glucose isomerase, which issensitive to Ca²⁺. Obviating Ca²⁺ removal saves time and cost andincreasing the efficiency of producing a high-fructose syrup.

Finally, AmyE polypeptides have a high activity towards ungelatinizedstarch, which can be recalcitrant to the enzymatic activity ofconventional α-amylases. This permits the use of jet cooked dry milledstarch for liquefaction and saccharification, where wet-milled starch isgenerally preferred to improve conversion efficiency.

It will be appreciated AmyE is suitable for use in aliquefaction/saccharification process that is tied to fermentation,isomerization, or any other subsequent process, including SSF.

5.1.6. Combination of AmyE with Glucoamylases and Other Enzymes

AmyE polypeptides can be used alone (e.g., as the only amylolytic enzymein starch processing) or can be combined with other α- or β-amylases, orother enzymes to provide a “cocktail” with a broad spectrum of activity.For example, the starch may be contacted with one or more enzymeselected from the group consisting of a fungal α-amylase (EC 3.2.1.1), abacterial α-amylase, e.g., a Bacillus α-amylase or a non-Bacillusα-amylase, or a β-amylase (EC 3.2.1.2). Another amylolytic enzyme or adebranching enzyme, such as an isoamylase (EC 3.2.1.68) or apullulanases (EC 3.2.1.41) may be combined with AmyE polypeptides.Isoamylase hydrolyses α-1,6-D-glucosidic branch linkages in amylopectinand β-limit dextrins and can be distinguished from pullulanases by theinability of isoamylase to attack pullulan and by the limited action ofisoamylase on α-limit dextrins.

β-Amylases are exo-acting maltogenic amylases, which catalyze thehydrolysis of 1,4-α-glucosidic linkages into amylose, amylopectin, andrelated glucose polymers, thereby releasing maltose. β-amylases havebeen isolated from various plants and microorganisms (Fogarty et al.,PROGRESS IN INDUSTRIAL MICROBIOLOGY, Vol. 15, pp. 112-115, 1979). Theseβ-amylases are characterized by having optimum temperatures in the rangefrom 40° C. to 65° C., and optimum pH in the range from about 4.5 toabout 7.0. Contemplated β-amylases include, but are not limited to,β-amylases from barley SPEZYME® BBA 1500, SPEZYME® DBA, OPTIMALT™ ME,OPTIMALT™ BBA (Danisco A/S); and NOVOZYM™ WBA (Novozymes A/S).

As described herein, AmyE polypeptides have glucoamylase activity andcan be used in the absence of a separate glucoamylase. Alternatively,glucoamylases may be added in a reduced amount compared to that requiredfor conventional stach hydrolysis methods. Preferably, glucoamlyses arepresent in an amount of no more than (i.e., less than) 0.5 glucoamylaseactivity unit (AGU)/g DS (i.e., glucoamylase activity units per gram ofdry solids), no more than 0.4 AGU/g DS, no more than 0.3 AGU/g DS, nomore than 0.2 AGU/g DS, or even no more than 0.1 AGU/g DS. Moregenerally, a glucoamylase may be added in an amount of 0.02-2.0 AGU/g DSor 0.1-1.0 AGU/g DS, although these ranges contemplate the use of moreglucoamylase than is required in combination with AmyE. Because AmyEpolypeptides are active at the same pH and temperature as glucoamylases,AmyE polypeptides may be added before or after addition of aglucoamylase, or simultaneously with a glucoamylase, e.g., by means of acocktail including both AmyE and a glucoamylase. Thus, the order andmanner of addition of an α-amylase and a glucoamylase are no longercritical, permitting increased flexibility in starch hydrolysisprocesses.

Glucoamylases (EC 3.2.1.3) may be derived from a microorganism or aplant. There are various known glucoamylases of fungal and bacterialorigin. Exemplary bacterial glucoamylases are Aspergillus glucoamylases,in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBOJ. 3:1097-1102), or variants thereof, such as disclosed in WO 92/00381and WO 00/04136; A. awamori glucoamylase (WO 84/02921); A. oryzaeglucoamylase (Agric. Biol. Chem. (1991) 55(4): 941-949), or variants orfragments thereof. Aspergillus glucoamylase variants include those thatenhance thermal stability: G137A and G139A (Chen et al. (1996) Prot.Eng. 9:499-505); D257E and D293E/Q (Chen et al. (1995) Prot. Eng.8:575-582); N182 (Chen et al. (1994) Biochem. J. 301:275-281);disulphide bonds, A246C (Fierobe et al. (1996) Biochemistry,35:8698-8704); and introduction of Pro residues in positions A435 andS436 (Li et al. (1997) Protein Eng. 10:1199-1204). Other glucoamylasesinclude Trichoderma reesie glucoamylase (e.g., SEQ ID NO: 3 of WO2006/060062; TrGA), Talaromyces glucoamylases, in particular derivedfrom T. emersonii (WO 99/28448), T. leycettanus (U.S. Pat. No. RE32,153), T. duponti, or T. thermophilus (U.S. Pat. No. 4,587,215).Bacterial glucoamylases include glucoamylases from the genusClostridium, in particular C. thermoamylolyticum (EP 135138) and C.thermohydrosulfuricum (WO 86/01831). Other suitable glucoamylasesinclude those derived from Aspergillus oryzae, such as a glucoamylasehaving 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or even 90% homology tothe amino acid sequence shown in SEQ ID NO: 2 in WO 00/04136. Alsosuitable are commercial glucoamylases, such as AMG 200L; AMG 300 L; SAN™SUPER and AMG™ E (Novozymes); OPTIDEX® 300 (Genencor Division, DaniscoUS Inc.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME® G900 (EnzymeBio-Systems); and G-ZYME® G990 ZR (A. niger glucoamylase and lowprotease content).

Phytases are enzymes capable of breaking down phytic acid (phytate)found in grains and oil seeds. Phytate, as well as intermediates in itdegredation, are believed to destabilize or otherwise adversely affectα-amylases, thereby reducing their efficiency. Phytases that can be usedin combination with variant α-amylases are capable of hydrolyzing phyticacid under the defined conditions of the incubation and liquefactionsteps. In some embodiments, the phytase is capable of liberating atleast one inorganic phosphate from an inositol hexaphosphate (phyticacid). Phytases can be grouped according to their preference for aspecific position of the phosphate ester group on the phytate moleculeat which hydrolysis is initiated, (e.g., as 3-phytases (EC 3.1.3.8) oras 6-phytases (EC 3.1.3.26)). A typical example of phytase ismyo-inositol-hexakiphosphate-3-phosphohydrolase.

Phytases can be obtained from microorganisms such as fungal andbacterial organisms. Some of these microorganisms include e.g.Aspergillus (e.g., A. niger, A. terreus, A. ficum and A. fumigatus),Myceliophthora (M. thermophila), Talaromyces (T. thermophilus)Trichoderma spp (T. reesei). and Thermomyces (WO 99/49740). Alsophytases are available from Penicillium species, e.g., P. hordei (ATCCNo. 22053), P. piceum (ATCC No. 10519), or P. brevi-compactum (ATCC No.48944). See, for example U.S. Pat. No. 6,475,762. In addition, phytasesare available from Bacillus (e.g., B. subtilis, Pseudomonas, Peniophora,E. coli, Citrobacter, Enterbacter and Buttiauxella (see WO2006/043178).

Commercial phytases are available such as NATUPHOS® (BASF), RONOZYME® P(Novozymes A/S), PHZYME® (Danisco A/S, Diversa) and FINASE® (ABEnzymes). The method for determining microbial phytase activity and thedefinition of a phytase unit has been published by Engelen et al. (1994)J. AOAC Int. 77:760-764. The phytase may be a wild-type phytase, avariant or fragment thereof.

Exemplary phytases are derived from species of the bacteriumButtiauxiella. Buttiauxiella spp. includes B. agrestis, B. brennerae, B.ferragutiase, B. gaviniae, B. izardii, B. noackiae, and B. warmboldiae.Strains of Buttiauxella species are available from DSMZ, the GermanNational Resource Center for Biological Material (Inhoffenstrabe 7B,38124 Braunschweig, DE). Buttiauxella sp. strain P1-29 deposited underaccession number NCIMB 41248 is an example of a particularly usefulstrain from which a phytase may be obtained. The phytase may be BP-wildtype, a variant thereof (such as BP-11) described in WO 06/043178, or avariant as described in U.S. Patent Pub. No. US20080220498, filed Mar.6, 2007 (see, e.g., Table 1 and SEQ ID NO: 3).

The phytase may also be the BP-17 variant of Buttiauxiella phytase,having the amino acid sequence of SEQ ID NO: 17, shown below, or aphytase having at least 75%, at least 80%, at least 85%, at least 88%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% and even at least99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17.

The amount (dosage) of phytase used in the incubation and/orliquefaction processes may be in the range of about 0.001 to 50 FTU/gds, (e.g., in the range of about 0.01 to 25 FTU/g ds, about 0.01 to 15FTU/g ds, about 0.01 to 10 FTU/g ds, about 0.05 to 15 FTU/g ds, andabout 0.05 to 5.0 FTU/g.

Other enzymes that may be used in combination with AmyE polypeptidesinclude lipases, cutinases, proteases, cellulases/hemicellulases,peroxidase, pectinase, pectine lyases, laccases, or combinations,thereof. In some cases a carbohydrate-binding domain of the typedisclosed in WO 98/22613 may be used.

5.2. Cleaning and Dishwashing Compositions and Methods

AmyE polypeptides can be formulated in detergent compositions for use incleaning dishes or other hard surfaces. These compositions can be gels,powders or liquids. The compositions may include AmyE polypeptides asthe single amylolytic enzymes in the composition. Alternatively, thecomposition may include other/additional amylolytic enzymes, othercleaning enzymes, and other components, many of which are common tocleaning compositions. The laundry detergent composition mayadditionally comprise one or more other enzymes, such as a lipase, acutinase, a protease, a cellulase, a peroxidase, a pectinase, a pectinelyase, and/or a laccase, or a combination, thereof.

Dishwashing detergent compositions generally include one or moresurfactants, which can be anionic, non-ionic, cationic, amphoteric or amixture of these types. The detergent can contain 0% to about 90% byweight of a non-ionic surfactant, such as low- to non-foamingethoxylated propoxylated straight-chain alcohols.

Liquid detergent compositions may include propylene glycol. AmyEpolypeptides can be solubilized in propylene glycol, for example, bycirculating in a 25% volume/volume propylene glycol solution containing10% calcium chloride.

Dishwashing detergent compositions may include detergent builder saltsof inorganic and/or organic types. Detergent builders may be subdividedinto phosphorus-containing and non-phosphorus-containing types.Detergent compositions usually contain about 1% to about 90% ofdetergent builders. Examples of phosphorus-containing inorganic alkalinedetergent builders are water-soluble salts, especially alkali metalpyrophosphates, orthophosphates, and polyphosphates. Examples ofphosphorus-containing organic alkaline detergent builders arewater-soluble salts of phosphonates. Examples ofnon-phosphorus-containing inorganic builders are water-soluble alkalimetal carbonates, borates, and silicates, as well as the various typesof water-insoluble crystalline or amorphous alumino silicates, of whichzeolites are the best-known representatives.

Examples of suitable organic builders are the alkali metal; ammonium andsubstituted ammonium; citrates; succinates; malonates; fatty acidsulphonates; carboxymethoxy succinates; ammonium polyacetates;carboxylates; polycarboxylates; aminopolycarboxylates; polyacetylcarboxylates; and polyhydroxsulphonates. Other suitable organic buildersinclude the higher molecular weight polymers and co-polymers known tohave builder properties, for example appropriate polyacrylic acid,polymaleic and polyacrylic/polymaleic acid copolymers, and their salts.

Cleaning compositions may contain bleaching agents, e.g., of thechlorine/bromine-type or the oxygen-type. Examples of inorganicchlorine/bromine-type bleaches are lithium, sodium or calciumhypochlorite, and hypobromite, as well as chlorinated trisodiumphosphate. Examples of organic chlorine/bromine-type bleaches areheterocyclic N-bromo- and N-chloro-imides such as trichloroisocyanuric,tribromoisocyanuric, dibromoisocyanuric, and dichloroisocyanuric acids,and salts thereof with water-solubilizing cations such as potassium andsodium. Hydantoin compounds are also suitable.

Cleaning compositions may contain oxygen bleaches, for example in theform of an inorganic persalt, optionally with a bleach precursor or as aperoxy acid compound. Typical examples of peroxy bleach compounds arealkali metal perborates, both tetrahydrates and monohydrates, alkalimetal percarbonates, persilicates, and perphosphates. Suitable activatormaterials include tetraacetylethylenediamine (TAED) and glyceroltriacetate. Enzymatic bleach activation systems may also be present,such as perborate or percarbonate, glycerol triacetate and perhydrolase,as disclosed in WO 2005/056783, for example.

Cleaning compositions may be stabilized using conventional stabilizingagents for the enzyme(s), e.g., a polyol such as, e.g., propyleneglycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boricacid derivative (e.g., an aromatic borate ester). The cleaningcomposition may also contain other conventional detergent ingredients,e.g., deflocculant material, filler material, foam depressors,anti-corrosion agents, soil-suspending agents, sequestering agents,anti-soil redeposition agents, dehydrating agents, dyes, bactericides,fluorescent agents, thickeners, and perfumes.

Finally, AmyE polypeptides may be used in conventional dishwashingdetergents, e.g., in any of the detergents described in the followingpatent publications, with the understanding that that AmyE polypeptidesare used instead of, or in addition to, the α-amylases described in thefollowing patents and published patent applications: CA 2006687, GB2200132, GB 2234980, GB 2228945, DE 3741617, DE 3727911, DE 4212166, DE4137470, DE 3833047, DE 4205071, WO 93/25651, WO 93/18129, WO 93/04153,WO 92/06157, WO 92/08777, WO 93/21299, WO 93/17089, WO 93/03129, EP481547, EP 530870, EP 533239, EP 554943, EP 429124, EP 346137, EP561452, EP 318204, EP 318279, EP 271155, EP 271156, EP 346136, EP518719, EP 518720, EP 518721, EP 516553, EP 561446, EP 516554, EP516555, EP 530635, EP 414197, and U.S. Pat. Nos. 5,112,518; 5,141,664;and 5,240,632.

AmyE polypeptide-containing detergent compositions may be formulated forhand or machine dishwashing operations.

5.3. Laundry Detergent Compositions and Methods

AmyE polypeptides may be a component of a laundry detergent composition,e.g., in the form of a non-dusting granulate, a stabilized liquid, aprotected enzyme, or the like. Non-dusting granulates may be produced,e.g., as described in U.S. Pat. Nos. 4,106,991 and 4,661,452 and mayoptionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products;(polyethyleneglycol, PEG) with mean molar weights of 1,000 to 20,000;ethoxylated nonylphenols having from 16 to 50 ethylene oxide units;ethoxylated fatty alcohols in which the alcohol contains from 12 to 20carbon atoms and in which there are 15 to 80 ethylene oxide units; fattyalcohols; fatty acids; and mono- and di- and triglycerides of fattyacids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are described in, e.g., GB PatentNo. 1,483,591.

Liquid enzyme preparations may be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods. Other enzyme stabilizers are known inthe art. Protected enzymes may be prepared according to the methoddisclosed in U.S. Pat. No. 5,879,920 (Danisco A/S) or EP 238216, forexample. Polyols have long been recognized as stabilizers of proteins aswell as for improving the solubility of proteins. See, e.g., Kaushik etal., J. Biol. Chem. 278: 26458-65 (2003) and references cited therein;and M. Conti et al., J. Chromatography 757: 237-245 (1997).

Laundry detergent composition may be in any convenient form, e.g., asgels, powders, granules, pastes, or liquids. A liquid detergent may beaqueous, typically containing up to about 70% of water, and 0% to about30% of organic solvent, it may also be in the form of a compact gel typecontaining only about 30% water.

Laundry detergent composition typically include one or more surfactants,which may be anionic, nonionic (including semi-polar), cationic, orzwitterionic, or a combination, thereof. The surfactants are typicallypresent at a level of from 0.1% to 60% by weight. In some cases, thedetergent will usually contain 0% to about 40% or to about 50% ofanionic surfactant, such as linear alkylbenzenesulfonate;α-olefinsulfonate; alkyl sulfate (fatty alcohol sulfate) (AS); alcoholethoxysulfate (AEOS or AES); secondary alkanesulfonates (SAS); α-sulfofatty acid methyl esters; alkyl- or alkenylsuccinic acid; or soap. Thecomposition may also contain 0% to about 40% of nonionic surfactant suchas alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide (as described in WO 92/06154), orN-acyl-N-alkyl derivatives of glucosamine (“glucamides”).

The laundry detergent composition may additionally comprise one or moreother enzymes, such as a lipase, a cutinase, a protease, a cellulase, aperoxidase, a pectinase, a pectine lyase, a laccase, and/or anotheramylolytic enzyme (e.g., another α-amylase), or a combination, thereof.In some cases, the 2,6-β-D-fructan hydrolase can be incorporated in alaundry detergent compositions and used for removal/cleaning of biofilmpresent on household and/or industrial textile/laundry.

The laundry detergent may contain about 1% to about 65% of a detergentbuilder or complexing agent such as zeolite, diphosphate, triphosphate,phosphonate, citrate, nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates orlayered silicates (e.g., SKS-6 from Hoechst). The detergent may also beunbuilt, i.e., essentially free of detergent builder. Enzymes may beused in any composition compatible with the stability of the enzyme.Enzymes can be protected against generally deleterious components byknown forms of encapsulation, as by granulation or sequestration inhydro gels, for example. Enzymes and specifically α-amylases either withor without the starch binding domains are not limited to laundry anddishwashing applications, but may bind use in surface cleaners andethanol production from starch or biomass.

The laundry detergent may comprise one or more polymers. Examplesinclude carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The laundry detergent may contain a bleaching system, which may comprisea H₂O₂ source such as perborate or percarbonate optionally combined witha peracid-forming bleach activator, such as TAED ornonanoyloxybenzenesulfonate (NOBS). Alternatively, the bleaching systemmay comprise peroxy acids of the amide, imide, or sulfone type, forexample. The bleaching system can also be an enzymatic bleaching systemwhere a perhydrolase activates peroxide, such as that described in WO2005/056783.

The enzymes of the laundry detergent composition, including AmyEpolypeptides may be stabilized using conventional stabilizing agents,e.g., a polyol such as propylene glycol or glycerol; a sugar or sugaralcohol; lactic acid; boric acid or a boric acid derivative, such as anaromatic borate ester; and the composition may be formulated asdescribed, e.g., in WO 92/19709 and WO 92/19708.

The laundry detergent may also contain other conventional detergentingredients such as fabric conditioners including clays, foam boosters,suds suppressors, anti-corrosion agents, soil-suspending agents,anti-soil redeposition agents, dyes, bactericides, optical brighteners,or perfume, for example. The pH (measured in aqueous solution at useconcentration) is usually neutral or alkaline, e.g., pH about 7.0 toabout 11.0, although AmyE polypeptides also work in low pH conditions,as in the case of starch hydrolysis.

One or more AmyE polypeptides may be present in laundry cleaningcompositions at concentrations conventionally employed in suchcompositions, e.g., 0.00001-1.0 mg (calculated as pure enzyme protein)of AmyE polypeptide per liter of wash liquor. Exemplary detergentcompositions include the following:

(1) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 7% to about 12%; alcohol ethoxysulfate (e.g.,C₁₂₋₁₈ alcohol, 1-2 ethylene oxide (EO)) or alkyl sulfate (e.g., C₁₆₋₁₈)about 1% to about 4%; alcohol ethoxylate (e.g., C₁₄₋₁₅ alcohol, 7 EO)about 5% to about 9%; sodium carbonate (e.g., Na₂CO₃) about 14% to about20%; soluble silicate, about 2 to about 6%; zeolite (e.g., NaAlSiO₄)about 15% to about 22%; sodium sulfate (e.g., Na₂SO₄) 0% to about 6%;sodium citrate/citric acid (e.g., C₆H₅Na₃O₇/C₆H₈O₇) about 0% to about15%; sodium perborate (e.g., NaBO₃.H₂O) about 11% to about 18%; TAEDabout 2% to about 6%; carboxymethylcellulose (CMC) and 0% to about 2%;polymers (e.g., maleic/acrylic acid, copolymer, PVP, PEG) 0-3%; enzymes(calculated as pure enzyme) 0.0001-0.1% protein; and minor ingredients(e.g., suds suppressors, perfumes, optical brightener, photobleach)0-5%.

(2) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 6% to about 11%; alcohol ethoxysulfate (e.g.,C₁₂₋₁₈ alcohol, 1-2 EO) or alkyl sulfate (e.g., C₁₆₋₁₈) about 1% toabout 3%; alcohol ethoxylate (e.g., C₁₄₋₁₅ alcohol, 7 EO) about 5% toabout 9%; sodium carbonate (e.g., Na₂CO₃) about 15% to about 21%;soluble silicate, about 1% to about 4%; zeolite (e.g., NaAlSiO₄) about24% to about 34%; sodium sulfate (e.g., Na₂SO₄) about 4% to about 10%;sodium citrate/citric acid (e.g., C₆H₅Na₃O₇/C₆H₈O₇) 0% to about 15%;carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,maleic/acrylic acid copolymer, PVP, PEG) 1-6%; enzymes (calculated aspure enzyme protein) 0.0001-0.1%; minor ingredients (e.g., sudssuppressors, perfume) 0-5%.

(3) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 5% to about 9%; alcohol ethoxylate (e.g.,C₁₂₋₁₅ alcohol, 7 EO) about 7% to about 14%; Soap as fatty acid (e.g.,C₁₆₋₂₂ fatty acid) about 1 to about 3%; sodium carbonate (as Na₂CO₃)about 10% to about 17%; soluble silicate, about 3% to about 9%; zeolite(as NaAlSiO₄) about 23% to about 33%; sodium sulfate (e.g., Na₂SO₄) 0%to about 4%; sodium perborate (e.g., NaBO₃.H₂O) about 8% to about 16%;TAED about 2% to about 8%; phosphonate (e.g., EDTMPA) 0% to about 1%;carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,maleic/acrylic acid copolymer, PVP, PEG) 0-3%; enzymes (calculated aspure enzyme protein) 0.0001-0.1%; minor ingredients (e.g., sudssuppressors, perfume, optical brightener) 0-5%.

(4) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 8% to about 12%; alcohol ethoxylate (e.g.,C₁₂₋₁₅ alcohol, 7 EO) about 10% to about 25%; sodium carbonate (asNa₂CO₃) about 14% to about 22%; soluble silicate, about 1% to about 5%;zeolite (e.g., NaAlSiO₄) about 25% to about 35%; sodium sulfate (e.g.,Na₂SO₄) 0% to about 10%; carboxymethylcellulose (CMC) 0% to about 2%;polymers (e.g., maleic/acrylic acid copolymer, PVP, PEG) 1-3%; enzymes(calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients(e.g., suds suppressors, perfume) 0-5%.

(5) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 21%;alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO or C₁₂₋₁₅ alcohol, 5 EO)about 12% to about 18%; soap as fatty acid (e.g., oleic acid) about 3%to about 13%; alkenylsuccinic acid (C₁₂₋₁₄) 0% to about 13%;aminoethanol about 8% to about 18%; citric acid about 2% to about 8%;phosphonate 0% to about 3%; polymers (e.g., PVP, PEG) 0% to about 3%;borate (e.g., B₄O₇) 0% to about 2%; ethanol 0% to about 3%; propyleneglycol about 8% to about 14%; enzymes (calculated as pure enzymeprotein) 0.0001-0.1%; and minor ingredients (e.g., dispersants, sudssuppressors, perfume, optical brightener) 0-5%.

(6) An aqueous structured liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 21%;alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO, or C₁₂₋₁₅ alcohol, 5 EO)3-9%; soap as fatty acid (e.g., oleic acid) about 3% to about 10%;zeolite (as NaAlSiO₄) about 14% to about 22%; potassium citrate about 9%to about 18%; borate (e.g., B₄O₇) 0% to about 2%; carboxymethylcellulose(CMC) 0% to about 2%; polymers (e.g., PEG, PVP) 0% to about 3%;anchoring polymers (e.g., lauryl methacrylate/acrylic acid copolymer);molar ratio 25:1, MW 3800) 0% to about 3%; glycerol 0% to about 5%;enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minoringredients (e.g., dispersants, suds suppressors, perfume, opticalbrighteners) 0-5%.

(7) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising fatty alcohol sulfate about 5% toabout 10%; ethoxylated fatty acid monoethanolamide about 3% to about 9%;soap as fatty acid 0-3%; sodium carbonate (e.g., Na₂CO₃) about 5% toabout 10%; soluble silicate, about 1% to about 4%; zeolite (e.g.,NaAlSiO₄) about 20% to about 40%; sodium sulfate (e.g., Na₂SO₄) about 2%to about 8%; sodium perborate (e.g., NaBO₃.H₂O) about 12% to about 18%;TAED about 2% to about 7%; polymers (e.g., maleic/acrylic acidcopolymer, PEG) about 1% to about 5%; enzymes (calculated as pure enzymeprotein) 0.0001-0.1%; and minor ingredients (e.g., optical brightener,suds suppressors, perfume) 0-5%.

(8) A detergent composition formulated as a granulate comprising linearalkylbenzenesulfonate (calculated as acid) about 8% to about 14%;ethoxylated fatty acid monoethanolamide about 5% to about 11%; soap asfatty acid 0% to about 3%; sodium carbonate (e.g., Na₂CO₃) about 4% toabout 10%; soluble silicate, about 1% to about 4%; zeolite (e.g.,NaAlSiO₄) about 30% to about 50%; sodium sulfate (e.g., Na₂SO₄) about 3%to about 11%; sodium citrate (e.g., C₆H₅Na₃O₇) about 5% to about 12%;polymers (e.g., PVP, maleic/acrylic acid copolymer, PEG) about 1% toabout 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; andminor ingredients (e.g., suds suppressors, perfume) 0-5%.

(9) A detergent composition formulated as a granulate comprising linearalkylbenzenesulfonate (calculated as acid) about 6% to about 12%;nonionic surfactant about 1% to about 4%; soap as fatty acid about 2% toabout 6%; sodium carbonate (e.g., Na₂CO₃) about 14% to about 22%;zeolite (e.g., NaAlSiO₄) about 18% to about 32%; sodium sulfate (e.g.,Na₂SO₄) about 5% to about 20%; sodium citrate (e.g., C₆H₅Na₃O₇) about 3%to about 8%; sodium perborate (e.g., NaBO₃.H₂O) about 4% to about 9%;bleach activator (e.g., NOBS or TAED) about 1% to about 5%;carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,polycarboxylate or PEG) about 1% to about 5%; enzymes (calculated aspure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., opticalbrightener, perfume) 0-5%.

(10) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 23%;alcohol ethoxysulfate (e.g., C₁₂₋₁₅ alcohol, 2-3 EO) about 8% to about15%; alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO, or C₁₂₋₁₅ alcohol,5 EO) about 3% to about 9%; soap as fatty acid (e.g., lauric acid) 0% toabout 3%; aminoethanol about 1% to about 5%; sodium citrate about 5% toabout 10%; hydrotrope (e.g., sodium toluensulfonate) about 2% to about6%; borate (e.g., B₄O₇) 0% to about 2%; carboxymethylcellulose 0% toabout 1%; ethanol about 1% to about 3%; propylene glycol about 2% toabout 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; andminor ingredients (e.g., polymers, dispersants, perfume, opticalbrighteners) 0-5%.

(11) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 20% to about 32%;alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO, or C₁₂₋₁₅ alcohol, 5 EO)6-12%; aminoethanol about 2% to about 6%; citric acid about 8% to about14%; borate (e.g., B₄O₇) about 1% to about 3%; polymer (e.g.,maleic/acrylic acid copolymer, anchoring polymer, such as laurylmethacrylate/acrylic acid copolymer) 0% to about 3%; glycerol about 3%to about 8%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%;and minor ingredients (e.g., hydrotropes, dispersants, perfume, opticalbrighteners) 0-5%.

(12) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising anionic surfactant (linearalkylbenzenesulfonate, alkyl sulfate, α-olefinsulfonate, α-sulfo fattyacid methyl esters, alkanesulfonates, soap) about 25% to about 40%;nonionic surfactant (e.g., alcohol ethoxylate) about 1% to about 10%;sodium carbonate (e.g., Na₂CO₃) about 8% to about 25%; solublesilicates, about 5% to about 15%; sodium sulfate (e.g., Na₂SO₄) 0% toabout 5%; zeolite (NaAlSiO₄) about 15% to about 28%; sodium perborate(e.g., NaBO₃.H₂O) 0% to about 20%; bleach activator (TAED or NOBS) about0% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%;minor ingredients (e.g., perfume, optical brighteners) 0-3%.

(13) Detergent compositions as described in compositions 1)-12) supra,wherein all or part of the linear alkylbenzenesulfonate is replaced by(C₁₂-C₁₈) alkyl sulfate.

(14) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising (C₁₂-C₁₈) alkyl sulfate about 9%to about 15%; alcohol ethoxylate about 3% to about 6%; polyhydroxy alkylfatty acid amide about 1% to about 5%; zeolite (e.g., NaAlSiO₄) about10% to about 20%; layered disilicate (e.g., SK56 from Hoechst) about 10%to about 20%; sodium carbonate (e.g., Na₂CO₃) about 3% to about 12%;soluble silicate, 0% to about 6%; sodium citrate about 4% to about 8%;sodium percarbonate about 13% to about 22%; TAED about 3% to about 8%;polymers (e.g., polycarboxylates and PVP) 0% to about 5%; enzymes(calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients(e.g., optical brightener, photobleach, perfume, suds suppressors) 0-5%.

(15) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising (C₁₂-C₁₈) alkyl sulfate about 4%to about 8%; alcohol ethoxylate about 11% to about 15%; soap about 1% toabout 4%; zeolite MAP or zeolite A about 35% to about 45%; sodiumcarbonate (as Na₂CO₃) about 2% to about 8%; soluble silicate, 0% toabout 4%; sodium percarbonate about 13% to about 22%; TAED 1-8%;carboxymethylcellulose (CMC) 0% to about 3%; polymers (e.g.,polycarboxylates and PVP) 0% to about 3%; enzymes (calculated as pureenzyme protein) 0.0001-0.1%; and minor ingredients (e.g., opticalbrightener, phosphonate, perfume) 0-3%.

(16) Detergent formulations as described in 1)-15) supra, which containa stabilized or encapsulated peracid, either as an additional componentor as a substitute for already specified bleach systems.

(17) Detergent compositions as described supra in 1), 3), 7), 9), and12), wherein perborate is replaced by percarbonate.

(18) Detergent compositions as described supra in 1), 3), 7), 9), 12),14), and 15), which additionally contains a manganese catalyst.

(19) Detergent composition formulated as a non-aqueous detergent liquidcomprising a liquid nonionic surfactant such as, e.g., linearalkoxylated primary alcohol, a builder system (e.g., phosphate), anenzyme(s), and alkali. The detergent may also comprise anionicsurfactant and/or a bleach system.

AmyE-polypeptide-containing laundry detergent compositions may beformulated as hand or machine laundry detergent compositions, includinglaundry additive compositions suitable for pre-treatment of stainedfabrics and rinse added fabric softener compositions, or may beformulated as detergent compositions for use in general household hardsurface cleaning operations.

The detergent compositions may include 2,6-β-D-fructan hydrolase, one ormore additional α-amylase enzymes, and one or more other cleaningenzymes, such as a protease, a lipase, a cutinase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, a laccase, and/or a peroxidase, and/orcombinations thereof. In general, the properties of the selectedenzyme(s) should be compatible with the selected detergent, (e.g.,pH-optimum, compatibility with other enzymatic and non-enzymaticingredients, etc.), and the enzyme(s) should be present in effectiveamounts.

A detergent additive, i.e., a separate additive or a combined additive,can be formulated as a granulate, liquid, slurry, etc. Suitablegranulate detergent additive formulations include non-dustinggranulates.

It is contemplated that in the detergent compositions, AmyE polypeptidesmay be added in an amount corresponding to about 0.01 to about 100 mg ofenzyme protein per liter of wash liquor, particularly about 0.05 toabout 5.0 mg of enzyme protein per liter of wash liquor, or even moreparticularly in 0.1 to about 1.0 mg of enzyme protein per liter of washliquor.

5.4. Enzymes for Use in Combination with AmyE Polypeptides

As described, above, AmyE polypeptide-containing cleaning compositionsmay include one or more additional enzymes, such as a protease, alipase, a cutinase, a carbohydrase, a cellulase, a pectinase, amannanase, an arabinase, a galactanase, a xylanase, an oxidase, alaccase, and/or a peroxidase, 2,6-β-D-fructan hydrolase, additionalα-amylase enzymes, and combinations thereof. In general, the propertiesof the selected enzyme(s) should be compatible with the selecteddetergent, (e.g., pH-optimum, compatibility with other enzymatic andnon-enzymatic ingredients, etc.), and the enzyme(s) should be present ineffective amounts. Exemplary enzymes are described, below. Many of theseenzymes can also be used in combination with AmyE polypeptides incompositions other than cleaning compositions.

Proteases: suitable proteases include those of animal, vegetable ormicrobial origin. Chemically modified or protein engineered mutants arealso suitable. The protease may be a serine protease or ametalloprotease, e.g., an alkaline microbial protease or a trypsin-likeprotease. Examples of alkaline proteases are subtilisins, especiallythose derived from Bacillus sp., e.g., subtilisin Novo, subtilisinCarlsberg, subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841),subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279). Examples oftrypsin-like proteases are trypsin (e.g., of porcine or bovine origin),and Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583).Examples of useful proteases also include but are not limited to thevariants described in WO 92/19729 and WO 98/20115. Suitable commerciallyavailable protease enzymes include ALCALASE®, SAVINASE®, PRIMASE™,DURALASE™, ESPERASE®, and KANNASE™ (Novo Nordisk A/S); MAXATASE®,MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT®, PURAFECT OXP™, FN2™, and FN3™(Danisco A/S).

Lipases: suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include, but are not limited to, lipases from Humicola(synonym Thermomyces), e.g. H. lanuginosa (T. lanuginosus) (see, e.g.,EP 258068 and EP 305216) and H. insolens (see, e.g., WO 96/13580); aPseudomonas lipase (e.g., from P. alcaligenes or P. pseudoalcaligenes;see, e.g., EP 218 272), P. cepacia (see, e.g., EP 331 376), P. stutzeri(see, e.g., GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705(see, e.g., WO 95/06720 and WO 96/27002), P. wisconsinensis (see, e.g.,WO 96/12012); a Bacillus lipase (e.g., from B. subtilis; see, e.g.,Dartois et al. Biochemica Biophysica Acta, 1131: 253-360 (1993)), B.stearothermophilus (see, e.g., JP 64/744992), or B. pumilus (see, e.g.,WO 91/16422). Additional lipase variants contemplated for use in theformulations include those described, for example, in: WO 92/05249, WO94/01541, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO95/14783, WO 95/22615, WO 97/04079, WO 97/07202, EP 407225, and EP260105. Some commercially available lipase enzymes include LIPOLASE® andLIPOLASE® Ultra (Novo Nordisk A/S).

Polyesterases: Suitable polyesterases include, but are not limited to,those described in WO 01/34899 (Danisco A/S) and WO 01/14629 (DaniscoA/S), and can be included in any combination with other enzymesdiscussed herein.

Amylases: The compositions can be combined with other α-amylases, suchas a non-variant α-amylase. These can include commercially availableamylases, such as but not limited to DURAMYL®, TERMAMYL™, FUNGAMYL® andBAN™ (Novo Nordisk A/S), RAPIDASE®, and PURASTAR® (Danisco A/S).

Cellulases: Cellulases can be added to the compositions. Suitablecellulases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Suitable cellulasesinclude cellulases from the genera Bacillus, Pseudomonas, Humicola,Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases producedfrom Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307; 5,648,263; 5,691,178;5,776,757; and WO 89/09259, for example. Exemplary cellulasescontemplated for use are those having color care benefit for thetextile. Examples of such cellulases are cellulases described in EP0495257; EP 531 372; WO 99/25846 (Danisco A/S), WO 96/34108 (DaniscoA/S), WO 96/11262; WO 96/29397; and WO 98/08940, for example. Otherexamples are cellulase variants, such as those described in WO 94/07998;WO 98/12307; WO 95/24471; PCT/DK98/00299; EP 531 315; U.S. Pat. Nos.5,457,046; 5,686,593; and 5,763,254. Commercially available cellulasesinclude CELLUZYME® and CAREZYME® (Novo Nordisk A/S); CLAZINASE™ andPURADAX® HA (Danisco A/S); and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases contemplated for usein the compositions include those of plant, bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful peroxidases include peroxidases from Coprinus, e.g., from C.cinereus, and variants thereof as those described in WO 93/24618, WO95/10602, and WO 98/15257. Commercially available peroxidases includeGUARDZYME™ (Novo Nordisk A/S), for example.

5.5. Assays for Measuring AmyE polypeptides Cleaning Performance

Exemplary assays for measuring AmyE polypeptides cleaning performanceare described below and in the appended Examples. One standard assaythat may be used to test the efficacy of a cleaning compositioncomprising an AmyE polypeptides involves a swatch test. Swatches havingstains of known “strength” on various types of material are commerciallyavailable (EMPA, St. Gallen, Switzerland; wfk—Testgewebe GmbH, KrefeldGermany; or Center for Test Materials, Vlaardingen, The Netherlands)and/or can be made by the practitioner (Morris and Prato, TextileResearch Journal 52(4): 280-286 (1982)). Swatches can comprise, forexample, a cotton-containing fabric containing a stain made byblood/milk/ink (BMI), spinach, grass, or chocolate/milk/soot. A BMIstain can be fixed to cotton with 0.0003% to 0.3% hydrogen peroxide, forexample. Other combinations include grass or spinach fixed with 0.001%to 1% glutaraldehyde, gelatin and Coomassie stain fixed with 0.001% to1% glutaraldehyde, or chocolate, milk and soot fixed with 0.001% to 1%glutaraldehyde.

The swatch can also be agitated during incubation with an AmyEpolypeptides and/or detergent formulation. Wash performance data isdependent on the orientation of the swatches in the wells (horizontalversus vertical), particularly in the 96-well plate. This would indicatethat mixing was insufficient during the incubation period. Althoughthere are a number of ways to ensure sufficient agitation duringincubation, a plate holder in which the microtiter plate is sandwichedbetween two plates of aluminum can be constructed. This can be as simpleas placing, for example, an adhesive plate sealer over the wells thenclamping the two aluminum plates to the 96-well plate with any type ofappropriate, commercially available clamps. It can then be mounted in acommercial incubator shaker. Setting the shaker to about 400 rpm resultsin very efficient mixing, while leakage or cross-contamination isefficiently prevented by the holder.

Trinitrobenzenesulfonic acid (TNBS) can be used to quantify theconcentration of amino groups in the wash liquor. This can serve as ameasure of the amount of protein that was removed from the swatch (see,e.g., Cayot and Tainturier, Anal. Biochem. 249: 184-200 (1997)).However, if a detergent or an enzyme sample leads to the formation ofunusually small peptide fragments (for example, from the presence ofpeptidases in the sample), then one will obtain a larger TNBS signal,i.e., more “noise.”

Another means of measuring wash performance of blood/milk/ink that isbased on ink release that can be quantified by measuring the absorbanceof the wash liquor. The absorbance can be measured at any wavelengthbetween 350 and 800 nm, e.g, 410 nm or 620 nm. The wash liquor can alsobe examined to determine the wash performance on stains containinggrass, spinach, gelatin or Coomassie stain. Suitable wavelengths forthese stains include and 670 nm for spinach or grass and 620 nm forgelatin or Coomassie. For example, an aliquot of the wash liquor(typically 100-150 μL from a 96-well microplate, for example) is removedand placed in a cuvette or multiwell microplate. This is then placed ina spectrophotometer and the absorbance is read at an appropriatewavelength. The system also can be used to determine a suitable enzymeand/or detergent composition for dish washing, for example, using ablood/milk/ink stain on a suitable substrate, such as cloth, plastic orceramic.

In one example, a BMI stain is fixed to cotton by applying 0.3% hydrogenperoxide to the BMI/cotton swatch for 30 minutes at 25° C. or byapplying 0.03% hydrogen peroxide to the BMI/cotton swatch for 30 minutesat 60° C. Smaller swatches of approximately 0.25″ are cut from theBMI/cotton swatch and placed in the wells of a 96-well microtiter plate.Into each well, a known mixture of a detergent composition and anenzyme, such as a variant protein, is placed. After placing an adhesiveplate sealer onto the top of the microtiter plate, the microtiter plateis clamped to an aluminum plate and agitated on an orbital shaker atapproximately 250 rpm for about 10 to 60 minutes. At the end of thistime, the supernatants are transferred to wells in a new microtiterplate and the absorbance of the ink at 620 nm is measured. This can besimilarly tests with spinach stains or grass stains fixed to cotton byapplying 0.01% glutaraldehyde to the spinach/cotton swatch orgrass/cotton swatch for 30 minutes at 25° C. The same can be done withchocolate, milk, and/or soot stains.

5.6. Textile Desizing Compositions and Methods

Also contemplated are compositions and methods of treating fabrics(e.g., to desize a textile) using one or more AmyE polypeptides. TheAmyE polypeptides can be used in any fabric-treating method known in theart (see, e.g., U.S. Pat. No. 6,077,316). For example, the feel andappearance of a fabric can be improved using a method involvingcontacting the fabric with an AmyE polypeptides in solution, optionallyunder pressure.

AmyE polypeptides may be applied during or after the weaving oftextiles, or during the desizing stage, or one or more additional fabricprocessing steps. During the weaving of textiles, the threads areexposed to considerable mechanical strain. Prior to weaving onmechanical looms, warp yarns are often coated with sizing starch orstarch derivatives in order to increase their tensile strength and toprevent breaking. AmyE polypeptides can be applied to remove thesesizing starch or starch derivatives. After the textiles have been woven,a fabric can proceed to a desizing stage. This can be followed by one ormore additional fabric processing steps. Desizing is the act of removingsize from textiles. After weaving, the size coating should be removedbefore further processing the fabric in order to ensure a homogeneousand wash-proof result. AmyE polypeptides may then be used in a methodfor desizing fabric, comprising enzymatic hydrolysis of the size by theaction of the AmyE polypeptides.

AmyE polypeptides can be used alone or with other desizing chemicalreagents and/or desizing enzymes to desize fabrics, includingcotton-containing fabrics. AmyE polypeptides can further be used incompositions and methods for producing a stonewashed look on indigo-dyeddenim fabric and garments. For the manufacture of clothes, the fabriccan be cut and sewn into clothes or garments, which are afterwardsfinished. In particular, for the manufacture of denim jeans, differentenzymatic finishing methods have been developed. The finishing of denimgarment normally is initiated with an enzymatic desizing step, duringwhich garments are subjected to the action of amylolytic enzymes toprovide softness to the fabric and make the cotton more accessible tothe subsequent enzymatic finishing steps. AmyE polypeptides can be usedin methods of finishing denim garments (e.g., a “bio-stoning process”),enzymatic desizing and providing softness to fabrics, and/or finishingprocess.

5.7. Compositions and Methods for Baking and Food Preparation

AmyE polypeptides may be used in compositions and methods for baking andfood preparation. For the commercial and home use of flour for bakingand food production, it is important to maintain an appropriate level ofα-amylase activity in the flour. A level of activity that is too highmay result in a product that is sticky and/or doughy and unmarketable.Conversely, flour with insufficient α-amylase activity may not containenough sugar for proper yeast function, resulting in dry, crumbly bread.Accordingly, AmyE polypeptides, alone or in combination with anotherα-amylase(s), may be added to the flour to augment the level ofendogenous α-amylase activity in flour. As described herein, AmyEpolypeptides have a temperature optimum in the range of 30-90° C.,50-80° C., 55-75° C., or even 60-70° C., which make them well-suited forbaking and food preparation applications. The temperature optimum ofdifferent AmyE variants may be measured, e.g., in a 1% solution ofsoluble starch at pH 5.5, or using other methods described herein orknown in the art.

In addition to the use of grains and other plant products in baking,grains such as barley, oats, and wheat, as well as plant components,such as corn, hops, and rice, are used for both commercial and homebrewing. The components used in brewing may be unmalted or may bemalted, i.e., partially germinated, resulting in an increase in thelevels of enzymes, including α-amylases. For successful brewing,adequate levels of α-amylase enzyme activity are necessary to ensure theappropriate levels of sugars for fermentation. AmyE polypeptides, aloneor in combination with another α-amylase(s), may be added to wort ormash to improve starch conversion. As described elsewhere herein, AmyEpolypeptides exhibit glucoamylase activity, allowing glucose to begenerated from starch-containing grains without the use of an additionalglucoamylase, or with a reduced amount of glucoamylase compared to thatrequired using other α-amylases.

AmyE polypeptides can further be added alone or in a combination withother amylases to prevent or retard the staling, i.e., crumb-firming, ofbaked products. The amount of AmyE polypeptide used for anti-stalingamylase will typically be in the range of 0.01-10 mg of enzyme proteinper kg of flour, e.g., 1-10 mg/kg. Additional anti-staling amylases thatcan be used in combination with AmyE polypeptides include anendo-amylase, e.g., a bacterial endo-amylase from Bacillus. Theadditional amylase can also be a maltogenic α-amylase (EC 3.2.1.133),e.g., from Bacillus. NOVAMYL® is a suitable maltogenic α-amylase from B.stearothermophilus strain NCIB 11837 and is described in Christophersenet al. (1997) Starch, 50:39-45. Other examples of anti-stalingendo-amylases include bacterial α-amylases derived from Bacillus, suchas B. licheniformis or B. amyloliquefaciens and exo-amylase, such asβ-amylase, e.g., from plant sources, such as soy bean, or from microbialsources, such as Bacillus.

Baking compositions comprising an AmyE polypeptides may further includea phospholipase. The phospholipase may have A₁ or A₂ activity to removefatty acid from the phospholipids, forming a lyso-phospholipid. Thephospholipase may or may not have lipase activity, i.e., activity ontriglycerides. It typically has a temperature optimum in the range of30-90° C., e.g., 30-70° C. The added phospholipases can be of animalorigin, for example, from pancreas, e.g., bovine or porcine pancreas,snake venom or bee venom. Alternatively, the phospholipase may be ofmicrobial origin, e.g., from filamentous fungi, yeast or bacteria, suchas the genus or species Aspergillus, A. niger; Dictyostelium, D.discoideum; Mucor, M. javanicus, M. mucedo, M. subtilissimus;Neurospora, N. crassa; Rhizomucor, R. pusillus; Rhizopus, R. arrhizus,R. japonicus, R. stolonifer; Sclerotinia, S. libertiana; Trichophyton,T. rubrum; Whetzelinia, W. sclerotiorum; Bacillus, B. megaterium, B.subtilis; Citrobacter, C. freundii; Enterobacter, E. aerogenes, E.cloacae; Edwardsiella, E. tarda; Etwinia, E. herbicola; Escherichia, E.coli; Klebsiella, K. pneumoniae; Proteus, P. vulgaris; Providencia, P.stuartii; Salmonella, S. typhimurium; Serratia, S. liquefasciens, S.marcescens; Shigella, S. flexneri; Streptomyces, S. violeceoruber;Yersinia, Y. enterocolitica; Fusarium, F. oxysporum, strain DSM 2672),for example.

A phospholipase may be added in an amount that improves the softness ofthe bread during the initial period after baking, particularly the first24 hours. The amount of phospholipase will typically be in the range of0.01-10 mg of enzyme protein per kg of flour, e.g., 0.1-5 mg/kg.Phospholipase activity will generally be in the range of 20-1,000 LipaseUnit (LU)/kg of flour, where a Lipase Unit is defined as the amount ofenzyme required to release 1 μmol butyric acid per minute at 30° C., pH7.0, with gum arabic as emulsifier and tributyrin as substrate.

Compositions of dough generally include wheat meal or wheat flour and/orother types of meal, flour or starch such as corn flour, cornstarch, ryemeal, rye flour, oat flour, oatmeal, soy flour, sorghum meal, sorghumflour, potato meal, potato flour or potato starch. The dough may befresh, frozen or par-baked. The dough can be a leavened dough or a doughto be subjected to leavening. The dough may be leavened in various ways,such as by adding chemical leavening agents, e.g., sodium bicarbonate orby adding a leaven, i.e., fermenting dough. Dough also may be leavenedby adding a suitable yeast culture, such as a culture of Saccharomycescerevisiae (baker's yeast), e.g., a commercially available strain of 1S.cerevisiae.

The dough may further comprise other conventional dough ingredients,e.g., proteins, such as milk powder, gluten, and soy; eggs (either wholeeggs, egg yolks or egg whites); an oxidant, such as ascorbic acid,potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammoniumpersulfate; an amino acid such as L-cysteine; a sugar; or a salt, suchas sodium chloride, calcium acetate, sodium sulfate or calcium sulfate.The dough may further comprise fat, e.g., triglycerides, such asgranulated fat or shortening, and/or an emulsifier such as mono- ordiglycerides, diacetyl tartaric acid esters of mono- or diglycerides,sugar esters of fatty acids, polyglycerol esters of fatty acids, lacticacid esters of monoglycerides, acetic acid esters of monoglycerides,polyoxyethylene stearates, or lysolecithin. The dough can also be madewithout the addition of an emulsifiers.

Optionally, an additional enzyme may be used together with theanti-staling amylase and the phospholipase. The additional enzyme may bea second (i.e., additional) amylase, such as an amyloglucosidase, aβ-amylase, a cyclodextrin glucanotransferase; a peptidase, in particularan exopeptidase; a transglutaminase; a lipase; a cellulase; ahemicellulase, in particular a pentosanase such as xylanase; a protease;a protein disulfide isomerase, e.g., a protein disulfide isomerase asdescribed in WO 95/00636; a glucanotranseferase; a branching enzyme(1,4-α-glucan branching enzyme); a 4-α-glucanotransferase (dextringlycosyltransferase); or an oxidoreductase, e.g., a peroxidase, alaccase, a glucose oxidase, a pyranose oxidase, a lipoxygenase, anL-amino acid oxidase or a carbohydrate oxidase. The additional enzymemay be of any origin, including mammalian and plant, and particularly ofmicrobial (bacterial, yeast or fungal) origin and may be obtained bytechniques conventionally used in the art.

The xylanase is typically of microbial origin, e.g., derived from abacterium or fungus, such as a strain of Aspergillus, in particular ofA. aculeatus, A. niger (e.g., WO 91/19782), A. awamori (e.g., WO91/18977), or A. tubigensis (e.g., WO 92/01793); from a strain ofTrichoderma, e.g., T. reesei, or from a strain of Humicola, e.g., H.insolens (e.g., WO 92/17573). PENTOPAN® and NOVOZYM 384® arecommercially available xylanase preparations produced from Trichodermareesei. The amyloglucosidase may be an A. niger amyloglucosidase (suchas AMG®). Other useful amylase products include GRINDAMYL® A 1000 or A5000 (available from Grindsted Products, Denmark) and AMYLASE® H orAMYLASE® P (available from Gist-Brocades, The Netherlands). The glucoseoxidase may be a fungal glucose oxidase, in particular an Aspergillusniger glucose oxidase (such as GLUZYME®). An exemplary protease isNeutrase®. An exemplary lipase can be derived from strains ofThermomyces (Humicola), Rhizomucor, Candida, Aspergillus, Rhizopus, orPseudomonas, in particular from Thermomyces lanuginosus (Humicolalanuginosa), Rhizomucor miehei, Candida antarctica, Aspergillus niger,Rhizopus delemar or Rhizopus arrhizus or Pseudomonas cepacia. The lipasemay be Lipase A or Lipase B derived from Candida antarctica as describedin WO 88/02775, for example, or the lipase may be derived fromRhizomucor miehei as described in EP 238,023, for example, or Humicolalanuginosa, described in EP 305,216, for example, or Pseudomonas cepaciaas described in EP 214,761 and WO 89/01032, for example.

The AmyE polypeptide-containing enzyme preparation may optionally in theform of a granulate or agglomerated powder. The preparation can have anarrow particle size distribution with more than 95% (by weight) of theparticles in the range from 25 to 500 μm. Granulates and agglomeratedpowders may be prepared by conventional methods, e.g., by spraying AmyEpolypeptides onto a carrier in a fluid-bed granulator. The carrier mayconsist of particulate cores having a suitable particle size. Thecarrier may be soluble or insoluble, e.g., a salt (such as NaCl orsodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol(such as sorbitol), starch, rice, corn grits, or soy.

Particles comprising AmyE polypeptides may also be encapsulated using afood grade lipid, which may be any naturally organic compound that isinsoluble in water but is soluble in non-polar organic solvents such ashydrocarbon or diethyl ether. Suitable food grade lipids include, butare not limited to, triglycerides either in the form of fats or oilswhich are either saturated or unsaturated. Examples of fatty acids andcombinations thereof which make up the saturated triglycerides include,but are not limited to, butyric (derived from milk fat), palmitic(derived from animal and plant fat), and/or stearic (derived from animaland plant fat). Examples of fatty acids and combinations thereof whichmake up the unsaturated triglycerides include, but are not limited to,palmitoleic (derived from animal and plant fat), oleic (derived fromanimal and plant fat), linoleic (derived from plant oils), and/orlinolenic (derived from linseed oil). Other suitable food grade lipidsinclude, but are not limited to, monoglycerides and diglycerides derivedfrom the triglycerides discussed above, phospholipids and glycolipids.

The food grade lipid, particularly in the liquid form, is contacted witha powdered form of AmyE polypeptides in such a fashion that the lipidmaterial covers (envelops) at least a portion of the surface of at leasta majority, e.g., 100% of the AmyE polypeptide particles. The advantagesof enveloping the AmyE polypeptide particles are two-fold. First, thefood grade lipid protects the enzyme from thermal denaturation duringthe baking process for those enzymes that are heat labile. Consequently,while the AmyE polypeptides are stabilized and protected during theproving and baking stages, they are released from the protective coatingin the final baked good product, where they hydrolyzes glucosidiclinkages in polyglucans. The loaded delivery vehicle also provides asustained release of the active enzyme into the baked good. That is,following the baking process, active AmyE polypeptides are continuallyreleased from the protective coating at a rate that counteracts, andtherefore reduces, the rate of staling.

In general, the amount of lipid applied to the AmyE polypeptideparticles can vary from a few percent of the total weight of theα-amylase to many times that weight, depending upon the nature of thelipid, the manner in which it is applied to the particles, thecomposition of the dough mixture to be treated, and the severity of thedough-mixing operation involved.

The lipid-enveloped enzyme is added to the ingredients used to prepare abaked good in an effective amount to extend the shelf-life of the bakedgood. The baker computes the amount of enveloped α-amylase, prepared asdiscussed above, that will be required to achieve the desiredanti-staling effect. The amount of the enveloped α-amylase required iscalculated based on the concentration of enzyme enveloped and on theproportion of α-amylase to flour specified. A wide range ofconcentrations has been found to be effective, although, as has beendiscussed, observable improvements in anti-staling do not correspondlinearly with the α-amylase concentration, but above certain minimallevels, large increases in α-amylase concentration produce littleadditional improvement. The α-amylase concentration actually used in aparticular bakery production could be much higher than the minimumnecessary in order to provide the baker with some insurance againstinadvertent under-measurement errors by the baker. The lower limit ofenzyme concentration is determined by the minimum anti-staling effectthe baker wishes to achieve.

A method of preparing a baked good may comprise: (a) preparinglipid-coated AmyE polypeptide particles, wherein substantially 100percent of the particles are coated; (b) mixing a dough containingflour; (c) adding the lipid-coated α-amylase to the dough before themixing is complete and terminating the mixing before the lipid coatingis removed from the α-amylase; (d) proofing the dough; and (e) bakingthe dough to provide the baked good, wherein the α-amylase is inactiveduring the mixing, proofing and baking stages and is active in the bakedgood. The enveloped AmyE polypeptides can be added to the dough duringthe mix cycle, e.g., near the end of the mix cycle to allow sufficientdistribution throughout the dough; however, the mixing stage isterminated before the protective coating becomes stripped from theparticle(s).

In some cases, bacterial α-amylase (BAA) can be added to thelipid-coated particles comprising AmyE polypeptides. BAA reduces breadto a gummy mass due to its excessive thermostability and retainedactivity in the fully baked loaf of bread; however, when BAA isincorporated into the lipid-coated particles, substantial additionalanti-staling protection is obtained, even at very low BAA dosage levels.

Various modifications and variation can be made to the compositions andmethods. All references cited herein are incorporated by reference intheir entirety for all purposes.

EXAMPLES

In the foregoing description and examples that follows, the followingabbreviations apply: wt % (weight percent); ° C. (degrees Centigrade);H₂O (water); dH₂O (deionized water); dIH₂O (deionized water, Milli-Qfiltration); g or gm (grams); μg (micrograms); mg (milligrams); kg(kilograms); μL and μl (microliters); mL and ml (milliliters); mm(millimeters); μm (micrometer); M (molar); mM (millimolar); μM(micromolar); U (units); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); DO (dissolved oxygen); W/V (weightto volume); W/W (weight to weight); V/V (volume to volume); IKA (IKAWorks Inc. 2635 North Chase Parkway SE, Wilmington, N.C.); Genencor(Danisco US Inc, Genencor Division, Palo Alto, Calif.); Ncm (Newtoncentimeter) and ETOH (ethanol). eq (equivalents); N (Normal); ds or DS(dry solids content), SAPU (spectrophotometric acid protease unit,wherein in 1 SAPU is the amount of protease enzyme activity thatliberates one micromole of tyrosine per minute from a casein substrateunder conditions of the assay) and GAU (glucoamylase unit, which isdefined as the amount of enzyme that will produce 1 g of reducing sugarcalculated as glucose per hour from a soluble starch substrate at pH 4.2and 60° C.).

Example 1 Plasmid Construction and Protein Expression

The following general method were used for plasmid construction andprotein expression.

1.1. Plasmid Construction

Nucleic acids encoding the AmyE of SEQ ID NO: 1 or a C-terminaltruncated AmyE variant, AmyE-tr (SEQ ID NO: 2), were cloned into the B.subtilis pHPLT expression vector, described in U.S. Pat. No. 5,024,943.FIG. 2 depicts the vector comprising a nucleic acid encoding AmyE-tr.

The pHPLT vector contained the B. licheniformis LAT promoter (“Plat”), asequence encoding the LAT signal peptide (“preLAT”), followed by PstIand Hpal restriction sites for cloning. “ori-pUB” is the origin ofreplication from pUB110; “reppUB” is the replicase gene from pUB110,“neo” is the neomycin/kanamycin resistance gene from pUB110; “bleo” isthe bleomycin resistance marker, “Tlat” is the transcriptionalterminator from B. licheniformis amylase. These and other features ofplasmid pUB110 are described in McKenzie et al., Plasmid 15(2): 93-103(1986).

Plasmid constructs for the expression of AmyE and AmyE-tr were assembledusing the AmyE-encoding sequence described by Yang et al, “Nucleotidesequence of the amylase gene from Bacillus subtilis,” Nucl. Acids Res.11(2): 237-49 (1983). Plasmid pME629.5 contains the nucleic acidencoding the full-length AmyE of SEQ ID NO: 1. The gene has a three basedeletion in the sequence encoding the starch binding domain, compared tothe sequence described by Yang et al.

Plasmid pME630.7, shown in FIG. 2, contained the truncated AmyE sequence(i.e., AmyE-tr). AmyE-tr is truncated at position D425 of SEQ ID NO: 1.AmyE-tr was designed based on a crystal structure of an AmyE variantthat lacked the starch binding domain, as described in Fujimoto et al.,“Crystal structure of a catalytic-site mutant alpha-amylase fromBacillus subtilis complexed with maltopentaose,” J. Mol. Biol. 277:393-407 (1998). See also, the RCSB Protein Data Bank© Accession No.1BAG, “Alpha-Amylase From Bacillus Subtilis Complexed WithMaltopentaose.”

For expression plasmid construction, the nucleic acid encoding the AmyEpolypeptide was PCR-amplified using HERCULASE® (Stratagene, La Jolla,Calif., USA) and purified using a column provided in a Qiagen QIAQUIK™PCR purification kit (Qiagen, Valencia, Calif., USA), and resuspended in50 μL of MILLI-Q™-purified water. 50 μL of the purified DNA was digestedsequentially with HpaI (Roche) and PstI (Roche), and the resultant DNAfragments was resuspended in 30 μL of MILLI-Q™-purified water. 10-20ng/μL DNA was cloned into plasmid pHPLT using the PstI and HpaI cloningsites. The ligation mixtures were directly transformed into competent B.subtilis cells (genotype: ΔaprE, ΔnprE, degUHy32 oppA, ΔspoIIE3501,amyE:xylRPxylAcomK-phleo). These B. subtilis cells had a competency gene(comK) placed under the control of a xylose-inducible promoter.Competency for DNA binding and uptake is induced by the addition ofxylose. Because the amyE gene in the parent plasmid has two PstI sites,a PCR fusion reaction was performed to remove these sites prior tocloning. PCR fusion was performed after two separate PCR reactions. Thefollowing primers were used for making the pHPLT construct using HpaIand PstI sites:

SEQ ID NO: 10: Primer PSTAMYE-F′ 5′-CTTCTTGCTGCCTCATTCTGCAGCTTCAGCACTT-ACAGCACCGTCGATCAAAAGCGGAAC-3′ SEQ ID NO: 11: Primer AMYENOPST-R′5′-CTGGAGGCACTATCCTGAAGGATTTCTCCGTATTG- GAACTCTGCTGATGTATTTGTG-3′SEQ ID NO: 12: Primer AMYENOPST-F′5′-CACAAATACATCAGCAGAGTTCCAATACGGAGAAA- TCCTTCAGGATAGTGCCTCCAG-3′SEQ ID NO: 13: Primer HPAIAMYE-R′5′-CAGGAAATCCGTCCTCTGTTAACTCAATGGGGAAGA- GAACCGCTTAAGCCCGAGTC-3′SEQ ID NO: 14: Primer HPAIAMYE-R′5′-CAGGAAATCCGTCCTCTGTTAACTCAATCAGGATAA- AGCACAGCTACAGACCTGG-3′SEQ ID NO: 15: Primer AMYE SEQ-F′ 5′-TACACAAGTACAGTCCTATCTG-3′SEQ ID NO: 16: Primer AMYE SEQ-F′ 5′-CATCCTCTGTCTCTATCAATAC-3′

The plasmids pME629.5 and pME630.7 express AmyE with a 31 residue signalsequence, which is cleaved post-translationally. The subsequent 10N-terminal amino acids are processed separately as proposed by Yang etal. (1983). pME629.5 encodes “full-length” AmyE and pME630.7 encodes“truncated” AmyE.

1.2. Protein Expression

Bacterial transformants harboring constructs encoding AmyE full-lengthand truncated polypeptides were selected on Luria agar (LA) with 10μg/mL neomycin, 1% insoluble starch and incubated overnight at 37° C.Transformants showing a clearing (or halo) around the colony wereselected for further studies. Precultures of each of the transformantswere grown for 8 h in LB with 10 μg/mL neomycin. 30 μL of eachpre-culture was added into a 250 mL flask filled with 30 mL ofcultivation media (described below) supplemented with 10 μg/mL neomycinand 5 mM CaCl₂. The cultivation media was an enriched semi-defined mediabased on MOPs buffer, with urea as the major nitrogen source, glucose asthe main carbon source, and supplemented with 1% soytone for robust cellgrowth. The shake flasks were incubated for 60-65 hours at 37° C., withmixing at 250 rpm. Cultures were harvested by centrifugation at 5,000rpm for 20 minutes in conical tubes. Since both AmyE full-length andAmyE truncated proteins expressed at high levels, the culturesupernatants were used for subsequent assays without furtherpurification.

Example 2 Common Assays

The following assays were used in the examples described below.Deviations from the protocols provided below are indicated in theindividual examples. In these experiments, a spectrophotometer was usedto measure the absorbance of the products formed after the completion ofthe reactions.

2.1. Amylase Activity Assay

Amylase activity was measured spectrophotometrically. Insoluble cornstarch covalently linked with the label Remazol Brilliant Blue R(“RBB-corn starch,” Sigma 57776) was used as a substrate. 75 μL of 2%(wt ds) slurry of RBB-corn starch in 50 mM sodium acetate, pH 4.5, 5.0or 5.6, was added to 10 μL of 100 μg/mL enzyme and thoroughly mixed. Themixture was then incubated at 50° C. for 30 minutes. The samples werethen placed on ice and substrate was removed by centrifugation at 4,100rpm for 20 minutes using a table-top centrifuge. The amount of productwas determined by measuring the amount of blue dye released from thestarch. The optical density (OD) of the dye was measured in triplicateat 595 nm.

2.2. Viscosity Measurement Assay

A viscometer was used to measure the viscosity of a corn starchsubstrate in the presence of an amylase at pH 4.5 and 5.8. A batch of30% ds corn starch substrate slurry was freshly made, using sulfuricacid to lower the pH to either 4.5 or 5.8. For each reaction, 50 g ofslurry (15 g ds) was weighed out and warmed for 10 minutes to 70° C.Upon addition of the α-amylase, the temperature was immediatelyincreased from 70° C. to 85° C., and the reaction was stirred at 75 rpm.Once the temperature of the slurry and enzyme mixture reached 85° C.,the viscosity was monitored for an additional 30 minutes.

2.3. Differential Scanning Calorimetry (DSC) to Measure ThermalStability

The excess heat capacity function of AmyE or a variant thereof wasmeasured in the presence or absence of 2 mM calcium chloride using anultrasensitive scanning high-throughput microcalorimeter (VP-CapillaryDSC; MicroCal, Inc., Northampton, Mass., USA). Approximately 500 μL of0.5 mg/mL of AmyE or a variant thereof were scanned over 30-120° C.temperature range. Truncated Geobacillus stearothermophilus α-amylase(AmyS) was used as a control. The amino acid sequence of AmyS, includinga 34 amino acid signal sequence, is shown in SEQ ID NO: 4. The samesample was then re-scanned to check the reversibility of the process.The buffer used was 10 mM sodium acetate, pH 5.5. A 200° C./hr scan ratewas used to minimize any artifacts resulting from aggregation. Thethermal midpoint (T_(m)) of the DSC curves was used as an indicator ofthermal stability. The standard error in all the T_(m) measurements wasless than 1%.

2.4. Bradford Assay in 96-Well Microtiter Plates

Protein concentration in sample supernatants was determined using theBradford QUICKSTART™ Dye Reagent (Bio-Rad, Hercules, Calif., USA).Samples were obtained by filtering broths from cultures grown inmicrotiter plates (MTPs) for 3 days at 37° C. with shaking at 280 rpmand humidified aeration. 10 μL of the culture filtrate was combined with200 μL Bradford QUICKSTART™ Dye Reagent in a well of a second MTP. Afterthorough mixing, the MTP were incubated for at least 10 minutes at roomtemperature. Air bubbles were removed and the OD (optical density) wasmeasured at 595 nm. To determine the protein concentration, thebackground reading (from uninoculated wells) was subtracted from thesample readings.

2.5. Glucose Formation by HPLC Measurement

Hydrolysis of Maltose and Maltoheptaose

0.5% maltose or maltoheptaose solutions were prepared in 50 mM sodiumacetate, pH 4.5 or 5.6, or in 50 mM malic acid pH 5.6, as specified foreach experiment. All enzyme samples were initially diluted to 1 mg/mL.Reaction mixtures were prepared by diluting the enzyme using theappropriate substrate solutions to give a final enzyme concentration of1 ppm, and then 200 μL aliquots were transferred to sterile screw toptubes and place in a 37° C. incubator. The reactions were stopped at theindicated times by diluting 10-fold into 10 mM sodium hydroxide.

Hydrolysis of Insoluble Starch

For measuring the hydrolysis of insoluble granular starch, purified AmyE or variants thereof (24.5 g/L) was diluted to a final concentration of20.4 ppm in malic acid buffer, pH 5.6. The protein was then added to a5% corn flour solution prepared in malic acid buffer, pH 5.6, to a finalconcentration of 1 ppm, and the mixture was incubated in a shaker at 32°C. Samples were periodically removed and diluted 10 fold into 50 mM NaOHto quench the reaction.

HPLC Detection Method

The formation of glucose and other breakdown products of the substrateswere analyzed by HPLC using an Agilent 1100 LC system equipped with aDionex PA-1 column and electrochemical detector. 10 μL samples wereinjected and a gradient of NaOH and sodium acetate was applied at 1.0mL/min at 25° C. The distribution of saccharides was determined frompreviously run standards. Elution profiles were obtained over 45minutes. Quantitation of glucose produced (reported as g/L) was obtainedusing authenticated glucose reference standard (Sigma, Mo., USA) toconvert peak area for the sugars to actual sugar concentrations.

2.6. Glucose Formation Using Maltose and Maltoheptaose Substrates

The breakdown of maltose or maltoheptaose to glucose was assayed byHPLC. 0.5% maltose and maltoheptaose solution were made in a 50 mMsodium acetate buffer at pH 4.5 and pH 5.6. All enzyme samples werediluted to 1 mg/mL from purified stocks. The 1 mg/mL enzyme sample wasfurther diluted into the maltose solution to give a final concentrationof 1 μg/mL enzyme. 200 μL aliquots were then added to sterile screw-toptubes and placed in a 37° C. incubator. The reactions were stopped after2, 5, and 8 days by the addition of sodium hydroxide. The formation ofglucose and the breakdown of maltose were analyzed by HPLC againstauthentic standards, using the methods described in Example 2.5.

2.7 Triplex Assay

Standard total reducing sugars, glucose, and iodine assays wereperformed separately to characterize the products of amylopectindigestion. A 2.2% (w/w) corn amylopectin substrate was prepared in 55 mMsodium acetate buffer pH 5.8 by boiling the mixture with stirring for 30minutes and then cooling to room temperature. One hundred μl ofsubstrate was placed in wells of a 96 well medium binding polystyrenemicrotiter plate and 10 μl of diluted culture supernatants of AmyEvariants were added to it. Plates were sealed (Nunc, cat. #236366) andimmediately placed in an iEMS shaking incubator and incubated at 50 Cfor 10 min, 1150 rpm. Amylase reactions were terminated via addition of20 μl 0.5 N NaOH with mixing.

Total reducing sugars present were determined by mixing 20 μl 5% w/v4-hydroxybenzhydrazide (Sigma H9882, prepared in 0.5 N HCl) with 80 μl0.5 N NaOH followed by 20 μl of amylase reaction in an full skirt PCRplate (Axygen PCR-96-FS-C). The plate was sealed (Microseal B adhesivesealer, BioRad MSB-1001) and incubated at 95 C for 2 minutes followed byimmediate cooling to 25 C on a PCR style thermocycling heating block. 80μl of reaction sample was transferred to a polystyrene 96 wellmicrotiter plate and the optical density was measured at 440 nm using aSpectramax plate reader.

Total glucose present in amylase reactions was determined in amicrotiter plate by mixing 20 μl of reaction sample with 50 μl 5.8 mg/ml2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt(Sigma, A1888) in 50 mM potassium phosphate buffer pH 6.8, containing0.005% v/v Tween 80 followed by addition of 30 μl solution containing0.33 U/ml peroxidase from horse radish type VI (Sigma, P8375), 3.33 U/mlOxyGo (Glucose Oxidase, Genencor) prepared in the same buffer. Themicrotiter plate was immediately placed in a Spectramax plate reader,shaken for 5 seconds and the optical density was monitored at 405 nm at9 second intervals for the time period of 60-180 seconds.

Iodine analysis of amylase reactions was performed by mixing a 1:4dilution (in water) of amylase reaction sample with 80 μl of Lugolsreagent (5 g iodine, 10 g potassium iodide dissolved in 100 ml water)diluted 1:20 in water in a polystyrene microtiter plate. Optical densityat 580 nm was determined using a Spectramax plate reader. MicrosoftExcel was used to assemble data acquired from the Softmax Pro software(plate reader).

Iodine results are reported as total OD580; chain length/residualamylose is a function of total OD, therefore amylase activity isinversely proportional to total OD. Total reducing sugars are reportedas total OD440 and are proportional to OD therefore amylase activity isdirectly proportional to OD. Total glucose is reported as a kinetic ratein the glucose assay and is directly proportional. OD and rates arereported in lieu of converting to known quantities using standard curvesconstructed from glucose. Ratios are reported using raw data and aretherefore unit-less. The three types of data were combined graphically,as a ratio of iodine divided by the ratio of total reducing sugars toglucose.

2.8 High-Throughput Viscometry Assay for Viscosity Reduction RateDetermination

A high-throughput viscometry assay was developed using the commerciallyavailable molecular rotor CCVJ (9-(2-carboxy-2-cyanovinyl) julolidine).A molecular rotor is a fluorescent species whose quantum yield (thenumber of photons emitted divided by the number of photons absorbed) isdependent on the free volume of the microenvironment, which is relatedin turn to viscosity. For such molecules, intramolecular rotation is thepreferred mode of relaxation from the excited state. Intramolecularrotation is inhibited in a manner proportionate to the viscosity of themicroenvironment, the balance of energy being dissipated throughradiative relaxation (fluorescent emission).

For measuring the rate of viscosity reduction due to enzymatic activity,the molecular rotor CCVJ was incorporated into a buffered suspension ofcorn amylopectin as follows. A 100 mM stock solution of CCVJ wasprepared by adding 186 μl of dimethyl sulfoxide to a vial containing 5mg of lyophilized CCVJ (Sigma Aldrich Corporation, St. Louis, Mo.). TheCCVJ stock solution was stored in the dark at room temperature andchecked for precipitation prior to use. 90 g of amylopectin from corn(MP Biomedicals LLC, Solon, Ohio) were added to 2,850 mL of distilledwater. This was heated to boiling with constant stiffing, under whichconditions the amylopectin gradually gelatinized and dissolved. Theresulting, uniformly-viscous suspension of gelled amylopectin wasremoved from the heat source and stirred continuously as it returned toroom temperature, at which point 150 mL of 1 M sodium acetate buffer pH5.8 (previously prepared by titrating 1 molar sodium acetate with 1 Macetic acid) were added, followed by 150 μl of Tween-80 (Sigma AldrichCorporation, St. Louis, Mo.). When the Tween-80 was completelydissolved, 150 μl of the 100 mM CCVJ stock solution were added anddissolved, at which point the amylopectin/CCVJ reagent was complete andready for use. The reagent was stored in clear glass at room temperaturewith constant stiffing for the three days that it took to complete theviscometry screen.

For the assay, all liquid-handling tasks were executed by a BiomekFX^(P) robot equipped with a multichannel head that enabled thesimultaneous pipetting of all 96 wells of a 96-well microtiter plate. 60μl of amylopectin/CCVJ reagent were added to each well of a blackuntreated polystyrene 96-well microtiter plate (Corning Incorporated,Corning, N.Y.). 30 μl of enzyme sample were pipetted on top of this andthe microtiter plate was immediately read on a Spectramax M2efluorometer (Molecular Devices Corporation, Sunnyvale, Calif.) that wasset up as follows: top-read fluorescence mode; excitation wavelength 435nanometers (nm); emission wavelength 495 nm; cutoff wavelength 455 nm;kinetic read mode with 24-second read interval; 15-second shake beforeinitial read, 3-second shake between reads; 192 seconds total read timewith a 20-second lag period (eliminating from each kinetic ratecalculation the first of the 9 data points collected).

In this assay, the rate of viscosity reduction was measured in terms ofthe rate of the drop in the fluorescent signal. Kinetic rates offluorescent signal reduction were automatically calculated as “Vmax(milli-units per min)” by Softmax Pro, the sofware that comes packagedwith Spectramax instruments.

2.9 High-Throughput Viscometry Assay for Post-Liquefaction ViscosityDetermination

For measuring the reduction in post-liquefaction viscosity due toenzymatic activity, the molecular rotor CCVJ was incorporated into abuffered slurry of corn flour as follows. A 100 mM stock solution ofCCVJ was prepared by adding 186 μl of dimethyl sulfoxide to a vialcontaining 5 mg of lyophilized CCVJ (Sigma Aldrich Corporation, St.Louis, Mo.). The CCVJ stock solution was stored in the dark at roomtemperature and checked for precipitation prior to use. Organic cornflour (Azure Farm, Dufur, Oreg.) was passed through a 600-micrometersieve, then baked at 95 degrees C. for 16 hours and returned to roomtemperature. A 20% (weight/weight), pH 5.8 corn flour slurry wasprepared in 2-kg batches by combining 1520 g of distilled water, 80 mLof 1 M sodium acetate buffer pH 5.6 (previously prepared by titrating 1molar sodium acetate with 1 molar acetic acid), 80 μl of Tween-80 (SigmaAldrich Corporation, St. Louis, Mo.), 80 μl of the 100 mM CCVJ stocksolution, and 400 g of sifted organic corn flour. The slurry was stirredvigorously and continuously for half an hour using a magnetic stir bar,at which point the pH was checked and confirmed to be 5.8. The slurrywas stirred vigorously and continuously to keep the corn flour uniformlydispersed, while 90 μl of slurry were added to each well of a blackuntreated polystyrene 96-well microtiter plate (Corning Incorporated,Corning, N.Y.) using a multichannel pipet with 200 μl tips trimmed to aninner diameter of approximately 2.3 millimeters, determined by verniercaliper (Mitutoyo Corporation, Kure, Hiroshima, Japan). For eachmicrotiter plate well, 10 μl of enzyme sample were pipetted on top ofthe corn flour slurry, after which the plate was sealed with an adhesivesealer (Bio-Rad Laboratories, Inc., Hercules, Calif.) and tightlysandwiched between pre-equilibrated metal plates in a high-temperatureoven set to 80 degrees C. The plate was incubated for one hour, afterwhich the plate was removed to room temperature and left overnight atroom temperature. The following day, the plate sealer was removed andthe plate was read on a Spectramax M2e fluorometer (Molecular DevicesCorporation, Sunnyvale, Calif.) that was set up as follows: top-readfluorescence mode; excitation wavelength 435 nanometers (nm); emissionwavelength 495 nm; cutoff wavelength 455 nm. In this assay, a decreasein fluorescent signal corresponded to a decrease in the density of thegelatinized corn flour, which in turn corresponded to an increase inliquefaction due to enzymatic activity.

Example 3 Specific Activity and pH Optima

The specific activity and pH optima of full-length AmyE (SEQ ID NO: 1),AmyE-tr (SEQ ID NO: 2), and variants, thereof, were measured usingassays described in Example 2.1. The specific activities were determinedby spectrophotometrically determining the amount of dye released from aRBB-corn starch substrate over 30 minutes at 50° C. and pH 4.5, 5.0, and5.6. FIG. 3 shows that AmyE-tr and Amy31A have highest specificactivities at pH 4.5, compared to pH 5 or 5.6. AmyE shows a higherspecific activity at pH 5 than at pH 5.6.

Example 4 Reduction of the Viscosity of a Corn Starch Substrate

The ability of AmyE, AmyE-tr, and variants, thereof, to reduce theviscosity of a corn starch substrate was determined using the assaymethod described in Example 2.2. The viscosity was measured as afunction of time at pH 4.5 and 5.8 for each enzyme. FIGS. 4A and 4B,respectively, show that AmyE-tr and AmyE reduced substrate viscosity atboth pH 4.5 and 5.8. The peak viscosities were the same, while a lowerfinal viscosity was observed at pH 5.8. FIGS. 4C and 4D, respectively,show that Amy31A has a better performance at pH 4.5 than pH 5.8.

Example 5 Thermal Stability

The thermal stabilities of AmyE, AmyE-tr, and variants, thereof, weremeasured in the presence and absence of Ca²⁺ to determine whether Ca²⁺contributed to the stability of the enzymes, using differential scanningcalorimetry (DSC) as described in Example 2.3. DSC revealed that thethermal unfolding process was irreversible. FIG. 5A shows the DSCunfolding profiles for AmyE and AmyE-tr, with and without 2 mM Ca²⁺.FIG. 5B shows the DSC unfolding profiles for full length Amy31A variant.The absence of any effect of calcium on the thermal melting pointssuggests that neither AmyE, AmyE-tr, nor Amy31A bind Ca²⁺ or arestabilized by Ca²⁺ in the mM concentration range. Contrasting resultswere obtained for G. stearothermophilus α-amylase (AmyS), as shown inFIG. 5C. Table 1 summarizes the melting temperature of the testedenzymes. Addition of Ca²⁺ did not significantly change the thermalstability of AmyE, AmyE-tr, or Amy31A, while Ca²⁺ significantlyincreased the stability of AmyS.

TABLE 1 Summary of DSC T_(m) measurements T_(m) (° C.) Sample Withoutadded CaCl₂ With 2 mM CaCl₂ ΔT_(m) AmyE truncated 74.6 74.5 −0.1 AmyEfull length 76.4 76 −0.4 Amy31A variant 74.8 74.4 −0.4 AmyS 100 107.37.3

Example 6 Conversion of Maltose and Maltoheptaose to Glucose

The ability of AmyE-tr (SEQ ID NO: 2) and six AmyE variants of positionQ153 (C, F, I, K, N, and V) of AmyE-tr to convert maltose andmaltoheptaose substrates to glucose at pH 5.6 was tested, using theglucose formation assay described in Example 2.3. Glucose generated wasmeasured after 1, 2, and 3 days. AmyE-tr and AmyE variants were used at1 ppm. Truncated AmyS (SEQ ID NO: 4) was used for comparison at asimilar dose.

FIG. 6 depicts the results of glucose production from a maltosesubstrate. The production of glucose by AmyS was minimal, while AmyE-trand the Q153 position variants produced significant amounts of glucose.The variant Q153N was the best producer of glucose under theseconditions. These results confirm that AmyE and a variant of AmyEefficiently produce glucose from maltose.

FIG. 7 depicts the results of glucose production from a maltoheptaosesubstrate. As was the case with maltose as a substrate, AmyS convertedmaltoheptaose to glucose with poor efficiency. By contrast, AmyE-tr andQ153 position variants converted maltoheptaose to glucose veryeffectively, with Q153K and F variants displaying the greatestconversion by day 3. This example demonstrates that AmyE and a variantof AmyE efficiently produce glucose from complex oligosaccharides.

Example 7 Conversion of Maltoheptaose to Glucose

The ability of AmyE-tr (SEQ ID NO: 2), AmyS, and AmyE variant Q153K toconvert maltoheptaose (DP7) to glucose (DP1) was tested. The products ofthe reactions were analyzed using the HPLC method described in Example2.2. Representative elution profiles are shown in FIGS. 8-10.

FIG. 8 shows that AmyE-tr converts DP7 predominately to DP1 and residualamounts of maltose (DP2) after a 72 h incubation. By contrast, FIG. 9shows that AmyS converts DP7 to smaller oligosaccharides over time,producing a mixture of DP5, DP4, DP3, DP2 and DP1 oligosaccharides. FIG.10 depicts a time course of the conversion of DP7 to smalleroligosaccharides in the presence of AmyE variant Q153K. Significantlevels of DP1, DP2, and DP3 were detected in as little as 1 h. By 3 h,the Q153K variant converted DP7 predominately to DP1. These results showthat AmyE and a variant thereof can efficiently produce glucose (DP1)from a DP7 substrate.

Example 8 Generation and Expression of Positional Variants

This example relates to the generation and expression of a library ofpositional variants.

8.1. Generation of Positional Libraries

Plasmid pME630.7 (FIG. 2, Example 1) was used to generate positionallibraries at 150 different amino acid residues of AmyE-tr. Table 1 listseach residue for which a positional library was made. Residues arenumbered based on their position in SEQ ID NO: 2. The amino acid listedat each position is the residue appearing in the AmyE of SEQ ID NO: 2(i.e., the wild type residue).

TABLE 1 SEQ ID Wild Variant NO: 2 type No. Numbering residue 1 1 L 2 2 T3 3 A 4 4 P 5 5 S 6 8 S 7 18 S 8 20 N 9 23 K 10 24 H 11 25 N 12 27 K 1328 D 14 30 H 15 35 T 16 44 Q 17 45 V 18 47 E 19 49 N 20 50 Q 21 51 G 2252 D 23 54 S 24 56 S 25 59 Y 26 68 Q 27 73 Y 28 75 G 29 76 T 30 78 Q 3185 A 32 88 E 33 89 E 34 90 Y 35 91 G 36 106 D 37 107 Y 38 108 A 39 109 A40 112 N 41 115 K 42 116 S 43 118 P 44 119 N 45 123 G 46 124 N 47 125 T48 126 Q 49 127 I 50 131 S 51 132 D 52 134 W 53 142 L 54 143 G 55 152 T56 153 Q 57 156 S 58 160 R 59 163 E 60 166 L 61 167 N 62 184 P 63 185 D64 187 G 65 188 S 66 190 G 67 192 Q 68 195 P 69 199 N 70 200 T 71 201 S72 202 A 73 203 E 74 212 D 75 213 S 76 214 A 77 218 A 78 219 A 79 221 A80 222 N 81 223 Y 82 233 H 83 234 S 84 238 A 85 240 K 86 241 N 87 243 N88 245 G 89 247 S 90 248 N 91 250 S 92 251 H 93 252 Y 94 253 A 95 254 S96 255 D 97 257 S 98 259 D 99 260 K 100 274 D 101 275 D 102 276 E 103277 E 104 282 S 105 283 D 106 284 D 107 287 R 108 307 P 109 308 E 110309 G 111 310 G 112 311 G 113 312 N 114 313 G 115 314 V 116 317 P 117318 G 118 319 K 119 320 S 120 321 Q 121 323 G 122 324 D 123 325 R 124327 S 125 328 A 126 331 E 127 333 Q 128 344 V 129 346 A 130 347 G 131349 H 132 357 G 133 358 N 134 359 N 135 367 G 136 368 S 137 369 H 138378 S 139 380 S 140 382 S 141 385 T 142 386 A 143 388 K 144 390 P 145393 R 146 395 D 147 400 A 148 401 G 149 402 S 150 406 N

The positional library for each of the 150 residues listed on Table 1contained approximately 16 amino acid substitution variants. Thelibraries consisted of transformed B. subtilis cells containingexpression plasmids encoding AmyE variant sequences at the 150 positionsdescribed. Each variant was confirmed by DNA sequencing analysis priorto protein activity evaluation. Individual clones were cultured asdescribed below to obtain the different AmyE variants for functionalcharacterization.

8.2. Protein Expression

The B. subtilis transformants containing AmyE substitution variants werecultured in 96 well plates for 8 hours in LB (Luria broth) with 10 μg/mlneomycin, and 30 μl of this pre-culture was added to a 250 mL flaskfilled with 30 mL of cultivation media (described below) supplementedwith 25 ppm chloramphenicol and 5 mM CaCl₂. The flasks were incubatedfor 60-65 hours at 37° C. with constant rotational mixing at 250 rpm.Cultures were harvested by centrifugation at 5,000 rpm for 20 minutes inconical tubes. The culture supernatants were used for assays. Thecultivation media was an enriched semi-defined media based on MOPsbuffer, with urea as major nitrogen source, glucose as the main carbonsource, and supplemented with 1% soytone for robust cell growth.

Example 9 Starch Hydrolysis Assay to Measure Specific Activity andThermal Stability

The AmyE position variants were evaluated using a starch hydrolysisassay to measure specific activity and thermal stability. AmyE variantsalso were assayed using a cleaning swatch assay to measure stain removalperformance. The pH stability of AmyE variants was evaluated bymeasuring amylase activity on a maltoheptaose substrate. Thermostabilityof each of the AmyE variants was determined by measuring amylaseactivity on a maltotriose substrate before and after heat stress.

9.1. Determination of Specific Activity and Thermal Stability

A starch hydrolysis assay was used to measure specific activity andstability of AmyE and AmyE variants. Conditions that closely mimicreal-world applications in cleaning and grain processing were used.Activity is defined as reducing ends generated by enzymatic breakdown ofcorn flour. Reducing ends were determined using a PAHBAH(p-hydroxybenzoic acid hydrazide) assay, described below. Stability isdefined as sustained activity at 80° C.

Hardware: Inheco Variomag Teleshake 95 heater shaker with PCR plateadapter (Hamilton Company, Reno Nev.); Thermo Electron Multidropautomated dispenser (Thermo Fisher Scientific, Inc., Waltham, Mass.);iEMS incubator (Thermo Fisher Scientific, Inc., Waltham, Mass.); V&PScientific stir disc dispenser (model VP722B); Axygen PCR-96-FS-Cfull-skirt PCR plate (Axygen Scientific, Inc., Union City, Calif.);Tetrad thermocyclers (MJ Research, Waltham, Mass.), Biomek® FX liquidhandlers (Beckman Coulter, Fullerton, Calif.).

Starch Hydrolysis: Azure Farms Organic Corn Flour (Norco, Calif., USA)was sifted to obtain the <600 micron fraction, baked 4 hours at 80° C.,then allowed to equilibrate overnight at room temperature. The prepareddry corn flour was delivered into Axygen PCR plates using the VP722Bunit as a powder flip dispenser. The mass of flour delivered to eachwell was determined to be approximately 5 mg. 100 μL 50 mM sodiumacetate pH 5.6 (for a final suspension pH of ˜5.8) were added to eachwell and mixed. Culture supernatants of AmyE and AmyE variants werediluted to approximately 20 μg/mL in dilution buffer (water+0.005%Tween-80). 10 μL diluted supernatant were transferred to 8-minute and30-minute reaction plates and mixed once by pipetting the sample up anddown. An aliquot of 50 μL light mineral oil was transferred to eachwell. Plates were transferred to Inheco units pre-heated to 80° C. Atvarious time points following incubation, the starch hydrolysis reactionwas stopped by addition of 10 μL of 4 N NaOH to each well. The starchhydrolysis reaction products were analyzed by the PAHBAH assay.

PAHBAH Assay: Aliquots of 80 μL of 0.5 N NaOH were added to all wells ofan empty PCR plate (a “PAHBAH reaction plate”), followed by 20 μL ofPAHBAH reagent (5% w/v p-hydroxybenzoic acid hydrazide (Sigma # H9882,St. Luois, Mo., USA), dissolved in 0.5 N HCl). The solutions were mixedby pipetting up and down. 10 μL of the starch hydrolysis reactionsupernatants were added to each well of the PAHBAH reaction plate. Theplates were sealed and placed in a thermocycler, programmed for 2minutes at 95° C., and then cooled to 20° C. Samples of 80 μL of thedeveloped PAHBAH reaction mixtures were transferred to a fresh plate,and absorbance was measured at 405 nm in a spectrophotometer.

9.2. Determination of Stain Removal Performance

The stain removal performance of AmyE and AmyE variants was determinedusing CS-28 rice starch stain microswatches. Microswatches of ¼-inchcircular diameter were obtained from CFT Vlaardingen (Netherlands). Twomicroswatches were placed into each well of a 96-well microtiter plate.

The filtered culture broth samples were tested at an appropriateconcentration by dilution with a mixture of 10 mM NaCl, 0.1 mM CaCl₂,0.005% Tween-80 to 20× the desired final concentration in theperformance test. The final concentration of enzyme was about 0.025-0.10ppm. Amylase performance was measured at both pH 8 and pH 10.

Either 190 μl of (A) a buffer solution containing 25 mM HEPES (Sigma,H7523), 2 mM CaCl₂, 0.005% Tween-80, pH 8.0, or (B) a buffer solutioncontaining 25 mM CAPS (Sigma, C2632), 2 mM CaCl₂, 0.005% Tween-80, pH10.0 were added to each well of the plates containing microswatches. 10μl of diluted amylase samples were added to each well to provide a totalvolume of 200 μl/well. The plate was covered with a plate seal andplaced in an iEMS incubator for 60 minutes at 40° C. with agitation at1,150 rpm. Following incubation under the appropriate conditions, 100 μlof solution from each well were removed and placed into a freshmicrotiter plate, and absorbance was measured at 488 nm in aspectrophotometer. “Blank controls,” containing 2 microswatches per welland detergent but no amylase samples, were also included in the test.

Calculation of the CS-28 rice starch hydrolysis performance: Theobtained absorbance value was corrected for the blank control value. Theresulting absorbance, “ΔOD488,” was a measure for the amylase activity.For each AmyE or AmyE variant, the performance index was calculated bydividing the activity of the variant by the activity of the wild-typeenzyme. The performance index thus represents a comparison of theperformance of the variant (actual value) and the standard AmyEreference enzyme (theoretical value) at the same protein concentration.In addition, the theoretical values were calculated, using theparameters of the Langmuir equation of the standard AmyE enzyme.

Variants with performance differences over the wild-type enzyme werecharacterized by a performance index (PI). A PI greater than 1 (PI>1)identified a better variant compared to the standard, e.g., wild-type,while a PI of 1 (PI=1) identified a variant that performs the same asthe standard. A PI less than 1 (PI<1) identified a variant that performsworse than the standard.

9.3. Determination of pH Stability

An amylase activity assay using maltoheptaose as a substrate was used todetermine pH stability of AmyE and AmyE variants. Alpha amylase activitywas measured by monitoring production of glucose at pH 5.8 and pH 4,using an enzyme-coupled colorimetric kinetic assay. Enzyme reactionswere carried out in flat-bottom polystyrene 96-well microtiter plates atroom temperature. For the assay conducted at pH 5.8, 5 μl of 5× dilutedculture supernatant of AmyE or AmyE variants in 0.005% (w/v) Tween-20 inwater were mixed with 45 μl of buffer containing sodium acetate, pH 5.8,CaCl₂, Tween-20, horseradish peroxidase (Sigma-Aldrich, cat. #8375) andglucose oxidase (OXYGO™; Genencor Division, Danisco US Inc.). The final50 μl volume contained 50 mM, 2.6 mM, 0.005% (w/v), 20 U/ml and 50 U/mlof each component, respectively. Reactions were initiated by theaddition of 50 μl of buffer containing 50 mM sodium acetate, pH 5.8, 5.4mg/ml 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammoniumsalt (Sigma-Aldrich, cat. # A1888) and 10 mM maltoheptaose(Sigma-Aldrich, cat. # M7753), and was followed by 5 seconds of mixing.Color formation in the reaction was monitored at 405 nm in 9 secondintervals for 240 seconds using a SpectraMAX 250 spectrophotometer(Molecular Devices, Union City, Calif.). Enzyme activity was reported asthe rate of color formation during the 120-240 second interval ofmonitoring. For the assay conducted at pH 4.0, the method as describedabove was repeated exactly except using buffer at pH 4.0 and 20 μl ofdiluted AmyE or AmyE variant samples with 30 μl of peroxidase/glucoseoxidase containing buffer, adjusting concentration of componentsappropriately.

9.4. Determination of Thermostability

The thermostability of AmyE and AmyE variants was measured bydetermining the amylase activity on maltotriose substrate at pH 5.8,monitoring the production of glucose using an enzyme-coupledcolorimetric kinetic assay. The assay method used here was the same asdescribed above using the maltoheptaose substrate. A 20 μl sample ofdiluted culture supernatants of AmyE or AmyE variants were used, andcolor formation was monitored during the 60-180 second interval of thereactions. In addition, 80 μl samples of diluted cultures were thentransferred to fresh plates, fitted with plate sealers, and incubatedfor 30 minutes at 60° C. with 650 rpm shaking on an iEMS device (ThermoFisher Scientific, Inc., Waltham, Mass.). Plates were cooled on ice for4 minutes then 20 μl samples were assayed for activity on maltotriosesubstrate as described above. As in the previous assay, for each AmyE orAmyE variant, the performance index was calculated by dividing theactivity of the variant by the activity of the wild-type enzyme. Theperformance index compared the performance of the variant (actual value)and the standard AmyE reference enzyme (theoretical value) at the sameprotein concentration.

Example 10 Relative Performance of AmyE Positional Variants

Using the procedures in Example 9, the relative performance or activityof AmyE-tr was compared to AmyE variants generated as described inExample 6. A total of 142 positional variants having six or more memberswere evaluated.

Definitions: The following definitions apply.

-   Performance Index (PI): ratio of performance of variant to parent    protein    -   Up mutations: PI>1    -   Neutral mutations: PI>0.5    -   Non-deleterious mutations: PI>0.05    -   Deleterious mutations: PI≦0.05-   Fully Restrictive Positions: No Neutral mutations for protein and    activity-   Non-Fully Restrictive Positions: At least one neutral mutation for    one of the properties tested-   Non-Restrictive Positions: ≧20% Neutral mutations for at least one    property

Table 2 summarizes the results of the site evaluation screens of AmyE-trand AmyE variants. In Table 2, Column 1 indicates the amino acidposition investigated. Column 2 shows the amino acid at that position inthe wild-type enzyme. Column 3 indicates the number of variants at thatposition that were investigated in this study. The subsequent columnsprovide the number of variants, followed by the percent (%) of Neutralmutations identified by each assay performed. The properties tested wereas follows: Columns 4 and 5 (corn flour ddG), specific activity on cornflour substrate; Columns 6 and 7 (DP3 ddG), maltotriose hydrolysis at pH5.8; Columns 8 and 9 (DP7 pH 4 ddG), maltoheptaose hydrolysis at pH 4;Columns 10 and 11 (DP7 pH 5.8 ddG) maltoheptaose hydrolysis at pH 5.8;Columns 12 and 13 (DP3 HS ddG), heat stability (30 min at 60° C.) usingmaltotriose hydrolysis assay; Columns 14 and 15 (Clean pH 8 ddG), ricestarch stain microswatch assay at pH 8; Columns 16 and 17 (Clean pH 10ddG), rice starch stain microswatch assay at pH 10. The 150 sitesevaluated in truncated AmyE contained two fully restrictive positions,i.e., 75 and 123. The 295 sites evaluated in full-length AmyE contained10 fully restrictive positions, i.e., 75, 97, 101, 102, 120, 133, 137,182, 266, and 306.

TABLE 2 Number and percentage of Neutral mutations (PI >0.5) at eachposition for each property tested. WT # at corn flour corn flour DP3 DP3DP7 pH4 DP7 pH4 DP7 pH5.8 DP7 pH5.8 DP3 HS DP3 HS Clean pH8 Clean CleanpH10 Clean pH10 Position amino acid Position ddG # ddG % ddG # ddG % ddG# ddG % ddG # ddG % ddG # ddG % ddG # pH8 ddG % ddG # ddG % 1 L 18 18100% 18 100% 18 100% 18 100% 17 94% 18 100% 18 100% 2 T 20 20 100% 20100% 20 100% 20 100% 18 90% 20 100% 20 100% 3 A 19 19 100% 19 100% 19100% 19 100% 19 100% 17 89% 19 100% 4 P 18 17 94% 18 100% 18 100% 18100% 16 89% 17 94% 18 100% 5 S 15 13 87% 14 93% 14 93% 14 93% 14 93% 1493% 13 87% 8 S 19 19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 19100% 18 S 12 12 100% 12 100% 12 100% 12 100% 12 100% 9 75% 10 83% 20 N19 19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 23 K 18 18100% 18 100% 18 100% 18 100% 18 100% 18 100% 17 94% 24 H 15 14 93% 15100% 15 100% 15 100% 15 100% 15 100% 14 93% 25 N 17 17 100% 17 100% 17100% 17 100% 17 100% 17 100% 17 100% 27 K 18 18 100% 18 100% 18 100% 18100% 18 100% 18 100% 17 94% 28 D 19 19 100% 19 100% 19 100% 19 100% 19100% 18 95% 19 100% 30 H 19 19 100% 19 100% 19 100% 19 100% 17 89% 19100% 19 100% 35 T 17 17 100% 17 100% 17 100% 17 100% 17 100% 17 100% 17100% 44 Q 20 18 90% 20 100% 20 100% 20 100% 20 100% 20 100% 20 100% 45 V14 14 100% 14 100% 13 93% 14 100% 10 71% 14 100% 7 50% 47 E 17 17 100%17 100% 17 100% 17 100% 17 100% 17 100% 17 100% 49 N 18 17 94% 18 100%18 100% 18 100% 18 100% 18 100% 15 83% 50 Q 19 19 100% 19 100% 19 100%19 100% 19 100% 17 89% 19 100% 51 G 19 12 63% 19 100% 19 100% 19 100% 1895% 19 100% 18 95% 52 D 20 19 95% 20 100% 19 95% 20 100% 16 80% 19 95%19 95% 54 S 18 18 100% 18 100% 17 94% 18 100% 17 94% 17 94% 17 94% 56 S18 18 100% 18 100% 18 100% 18 100% 18 100% 18 100% 18 100% 59 Y 17 7 41%17 100% 10 59% 8 47% 15 88% 17 100% 3 18% 68 Q 19 19 100% 19 100% 19100% 19 100% 19 100% 19 100% 18 95% 73 Y 15 5 33% 14 93% 8 53% 14 93% 1067% 14 93% 13 87% 76 T 15 14 93% 14 93% 15 100% 15 100% 15 100% 13 87%11 73% 78 Q 19 19 100% 19 100% 18 95% 18 95% 19 100% 19 100% 19 100% 85A 15 15 100% 15 100% 15 100% 15 100% 15 100% 15 100% 15 100% 88 E 19 19100% 19 100% 19 100% 18 95% 18 95% 18 95% 17 89% 89 E 20 20 100% 20 100%20 100% 20 100% 20 100% 20 100% 20 100% 90 Y 18 18 100% 18 100% 18 100%18 100% 18 100% 18 100% 18 100% 91 G 18 13 72% 17 94% 17 94% 17 94% 1794% 15 83% 15 83% 106 S 18 9 50% 18 100% 9 50% 18 100% 6 33% 18 100% 1478% 107 Y 19 14 74% 19 100% 19 100% 19 100% 13 68% 19 100% 17 89% 108 A18 18 100% 18 100% 18 100% 18 100% 18 100% 18 100% 18 100% 109 A 18 1689% 18 100% 18 100% 17 94% 17 94% 18 100% 15 83% 112 N 20 20 100% 19 95%20 100% 20 100% 18 90% 20 100% 20 100% 115 K 18 14 78% 18 100% 18 100%18 100% 14 78% 15 83% 12 67% 116 S 16 15 94% 16 100% 16 100% 16 100% 16100% 16 100% 16 100% 118 P 20 18 90% 20 100% 20 100% 20 100% 20 100% 20100% 19 95% 119 N 16 15 94% 16 100% 16 100% 16 100% 13 81% 16 100% 1594% 124 N 15 1 7% 15 100% 7 47% 15 100% 1 7% 14 93% 10 67% 125 T 18 844% 18 100% 18 100% 18 100% 7 39% 17 94% 16 89% 126 Q 17 9 53% 16 94% 1482% 16 94% 13 76% 16 94% 12 71% 131 S 17 15 88% 17 100% 17 100% 17 100%15 88% 17 100% 16 94% 132 D 16 2 13% 16 100% 15 94% 14 88% 2 13% 14 88%6 38% 134 W 14 0 0% 14 100% 14 100% 14 100% 0 0% 13 93% 12 86% 142 L 156 40% 15 100% 3 20% 3 20% 13 87% 9 60% 3 20% 143 G 13 1 8% 13 100% 2 15%13 100% 1 8% 2 15% 2 15% 152 T 20 20 100% 20 100% 20 100% 20 100% 19 95%20 100% 20 100% 153 Q 20 19 95% 19 95% 19 95% 19 95% 19 95% 19 95% 1995% 156 S 19 19 100% 19 100% 19 100% 19 100% 18 95% 19 100% 17 89% 160 R19 19 100% 19 100% 19 100% 19 100% 19 100% 18 95% 19 100% 163 D 18 18100% 18 100% 18 100% 18 100% 18 100% 18 100% 18 100% 166 L 19 19 100% 19100% 18 95% 19 100% 19 100% 19 100% 18 95% 167 N 18 18 100% 18 100% 18100% 18 100% 18 100% 18 100% 18 100% 184 P 19 2 11% 19 100% 19 100% 19100% 1 5% 17 89% 7 37% 185 D 6 2 33% 6 100% 6 100% 6 100% 1 17% 6 100% 350% 187 G 20 1 5% 20 100% 20 100% 20 100% 2 10% 19 95% 19 95% 188 S 17 953% 17 100% 17 100% 17 100% 10 59% 17 100% 16 94% 190 G 13 6 46% 13 100%13 100% 13 100% 2 15% 13 100% 10 77% 195 P 18 4 22% 18 100% 18 100% 18100% 6 33% 16 89% 17 94% 199 N 17 15 88% 17 100% 16 94% 16 94% 17 100%14 82% 11 65% 200 T 20 18 90% 19 95% 19 95% 19 95% 14 70% 18 90% 17 85%201 S 20 20 100% 20 100% 20 100% 20 100% 20 100% 20 100% 20 100% 202 A16 13 81% 16 100% 16 100% 16 100% 15 94% 14 88% 14 88% 203 E 17 17 100%17 100% 17 100% 17 100% 17 100% 17 100% 17 100% 212 D 12 1 8% 12 100% 542% 12 100% 1 8% 6 50% 5 42% 213 S 18 15 83% 18 100% 18 100% 17 94% 1794% 15 83% 16 89% 214 A 17 6 35% 17 100% 17 100% 17 100% 10 59% 17 100%15 88% 218 A 20 20 100% 20 100% 20 100% 20 100% 18 90% 20 100% 20 100%219 A 18 6 33% 18 100% 18 100% 18 100% 6 33% 17 94% 7 39% 221 A 14 1071% 14 100% 13 93% 14 100% 8 57% 12 86% 11 79% 233 H 20 18 90% 20 100%19 95% 20 100% 19 95% 20 100% 9 45% 234 S 19 19 100% 19 100% 19 100% 19100% 18 95% 19 100% 19 100% 238 A 19 18 95% 19 100% 19 100% 19 100% 19100% 18 95% 19 100% 240 K 18 18 100% 18 100% 18 100% 18 100% 18 100% 18100% 17 94% 241 N 18 18 100% 18 100% 18 100% 18 100% 18 100% 18 100% 18100% 243 N 19 19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 19 100%245 G 16 16 100% 16 100% 16 100% 16 100% 16 100% 15 94% 16 100% 247 S 1614 88% 14 88% 14 88% 14 88% 14 88% 14 88% 14 88% 250 S 19 18 95% 19 100%18 95% 19 100% 18 95% 18 95% 19 100% 251 H 16 16 100% 16 100% 16 100% 16100% 16 100% 16 100% 16 100% 252 Y 18 14 78% 18 100% 16 89% 18 100% 950% 14 78% 17 94% 253 A 17 17 100% 17 100% 17 100% 17 100% 15 88% 17100% 16 94% 254 S 15 12 80% 13 87% 13 87% 15 100% 9 60% 15 100% 12 80%259 D 16 15 94% 16 100% 15 94% 15 94% 16 100% 15 94% 14 88% 260 K 19 19100% 19 100% 19 100% 19 100% 19 100% 16 84% 18 95% 274 D 19 18 95% 19100% 1 5% 19 100% 19 100% 19 100% 19 100% 275 D 20 19 95% 20 100% 8 40%20 100% 20 100% 20 100% 18 90% 276 E 20 19 95% 18 90% 14 70% 18 90% 1890% 19 95% 16 80% 277 E 20 19 95% 20 100% 10 50% 20 100% 18 90% 20 100%15 75% 282 S 20 18 90% 20 100% 19 95% 20 100% 20 100% 20 100% 20 100%283 D 17 14 82% 16 94% 7 41% 16 94% 16 94% 15 88% 16 94% 284 D 20 19 95%19 95% 19 95% 19 95% 19 95% 19 95% 20 100% 287 R 20 18 90% 20 100% 20100% 20 100% 20 100% 18 90% 20 100% 307 P 6 4 67% 6 100% 3 50% 6 100% 6100% 6 100% 6 100% 308 E 20 20 100% 20 100% 20 100% 20 100% 20 100% 20100% 19 95% 309 G 17 14 82% 16 94% 11 65% 16 94% 16 94% 11 65% 17 100%310 G 17 5 29% 17 100% 2 12% 17 100% 16 94% 15 88% 14 82% 311 G 20 1890% 19 95% 2 10% 20 100% 19 95% 17 85% 19 95% 312 N 20 19 95% 20 100% 1995% 19 95% 20 100% 20 100% 18 90% 313 G 20 17 85% 20 100% 17 85% 20 100%20 100% 20 100% 20 100% 314 V 16 15 94% 15 94% 15 94% 15 94% 15 94% 16100% 15 94% 317 P 19 19 100% 19 100% 14 74% 19 100% 19 100% 19 100% 1789% 318 G 16 15 94% 15 94% 15 94% 15 94% 15 94% 14 88% 15 94% 319 K 1212 100% 12 100% 12 100% 12 100% 12 100% 12 100% 12 100% 320 S 18 18 100%18 100% 13 72% 18 100% 18 100% 18 100% 18 100% 321 Q 18 17 94% 18 100%17 94% 18 100% 18 100% 18 100% 18 100% 323 G 7 6 86% 6 86% 2 29% 6 86% 571% 3 43% 7 100% 324 D 20 19 95% 20 100% 19 95% 20 100% 20 100% 18 90%20 100% 325 R 17 15 88% 17 100% 16 94% 17 100% 17 100% 14 82% 17 100%327 S 13 12 92% 11 85% 6 46% 11 85% 11 85% 8 62% 12 92% 328 A 19 19 100%19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 331 E 16 16 100% 16 100%16 100% 16 100% 16 100% 16 100% 16 100% 333 Q 18 18 100% 18 100% 18 100%18 100% 18 100% 18 100% 18 100% 344 V 18 15 83% 18 100% 18 100% 18 100%18 100% 18 100% 18 100% 346 A 16 16 100% 16 100% 16 100% 16 100% 16 100%16 100% 16 100% 347 G 20 20 100% 20 100% 20 100% 20 100% 20 100% 20 100%20 100% 349 P 19 19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 19 100%357 G 20 20 100% 20 100% 20 100% 20 100% 20 100% 20 100% 20 100% 358 N19 19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 18 95% 359 N 20 20100% 20 100% 20 100% 20 100% 20 100% 20 100% 20 100% 367 G 19 19 100% 19100% 19 100% 19 100% 19 100% 19 100% 19 100% 368 S 16 16 100% 16 100% 16100% 16 100% 16 100% 16 100% 16 100% 369 H 20 20 100% 20 100% 20 100% 20100% 20 100% 20 100% 20 100% 378 S 20 20 100% 20 100% 20 100% 20 100% 20100% 20 100% 20 100% 380 S 18 18 100% 18 100% 18 100% 18 100% 18 100% 18100% 18 100% 382 S 19 19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 19100% 385 T 20 20 100% 20 100% 20 100% 20 100% 20 100% 20 100% 20 100%386 A 16 16 100% 16 100% 16 100% 16 100% 16 100% 16 100% 15 94% 388 K 1919 100% 19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 390 P 19 19 100%19 100% 19 100% 19 100% 19 100% 19 100% 19 100% 393 R 17 17 100% 17 100%17 100% 17 100% 17 100% 17 100% 17 100% 395 D 20 20 100% 19 95% 19 95%20 100% 19 95% 20 100% 20 100% 400 A 20 20 100% 20 100% 20 100% 20 100%20 100% 20 100% 20 100% 401 G 20 20 100% 20 100% 20 100% 20 100% 20 100%20 100% 20 100% 402 S 18 18 100% 18 100% 18 100% 18 100% 18 100% 18 100%18 100% 406 N 6 6 100% 6 100% 6 100% 6 100% 6 100% 6 100% 6 100%

Example 11 Ethanol Formation by AmyE

In this example, experiments were conducted to test the performance oftruncated AmyE in conventional ethanol fermentation on Illinois RiverEnergy (IRE) liquefact (31% DS) using a conventional ethanolfermentation assay. Briefly, batches of 31% DS Illinois River Energy(IRE) liquefact with 400 ppm urea were prepared and the pH of one batchadjusted to 4.3 and the other adjusted to pH 5.8 with 5 N H₂SO₄. 100 gsubstrate was used per flask (125 ml Erlenmeyer). Enzymes were dosed asindicated. Fermentations were inoculated with 0.2 ml of 10% (w/v) RedStar Ethanol Red yeast (prehydrated ˜45 min in DI water). Flasks wereincubated at 32° C. with stir bars at 320 rpm for 48 h fermentation. Theamount of ethanol produced was measured by HPLC analysis. Theperformance of truncated AmyE (SEQ ID NO: 2) was compared to SPEZYME®Xtra amylase (SEQ ID NO: 4) at pH 4.3 and pH 5.8. Truncated AmyE andSPEZYME® Xtra amylase were dosed at 0.2 mg/gDS.

As shown in FIG. 11, the final ethanol yield produced by truncated AmyEat pH 5.8 is 12.0% (v/v). Truncated AmyE at pH 4.3 yielded a finalethanol yield of 7.3% (v/v). Final ethanol yields in the presence ofSPEZYME® Xtra amylase were 2.7% (v/v) at pH 4.3 and 3.9% (v/v) at pH5.8.

Example 12 Comparison of Ethanol Formation by AmyE and Other α-Amylases

In this example, experiments were conducted to compare the ability offull length Amy E (SEQ ID NO: 1) and truncated AmyE (SEQ ID NO: 2) tohydrolyze insoluble granular (uncooked) starch into ethanol at pH 4.3and pH 5.8, using the ethanol fermentation on whole ground corn assaydescribed in Example 11.

Using this assay, the ethanol forming performance of full length andtruncated AmyE was compared to A. kawachii alpha amylase (AkAA, GC626),dosed at 1.5 SSU/g (one unit of the enzyme activity-SSU soluble starchunit is equivalent to the reducing power of 1 mg of glucose released perminute from the hydrolyssi of soluble potato starch substrate (4% ds) atpH 4.5 and 50° C.) and STARGEN™ 002 (Aspergillus kawachi alpha amylaseexpressed in Trichoderma reesei and a glucoamylase from Trichodermareesei that work synergistically to hydrolyze granular starch substrateto glucose), dosed at 0.5 GAU/g, where one Glucoamylase Unit (GAU) isthe amount of enzyme that will produce 1 μM of reducing sugar,calculated as glucose per hour from a soluble starch substrate (4% ds)at pH 4.2 and 60° C. The definitions of SSU and GAU are described ingreater detail in U.S. Pat. No. 7,037,704. Both, AmyE full-length andtruncated AmyE were dosed at 0.2 mg/gDS.

FIG. 12 shows the results observed when performance of these enzymeswere compared, at pH 4.3 and 5.8, and reported as final ethanol yieldproduced. When tested at pH 5.8, both full length and truncated AmyEperformed very comparably to the STARGEN™ 002, with full length AmyEactually surpassing the ethanol yields observed for Stargen™ 002 at pH4.3. The truncated AmyE when tested at pH 5.8 performed very comparablyto Stargen™ 002 tested at the same pH. In comparison, the A. kawachiialpha amylase performed very poorly at both pH 4.2 and pH 5.8.

Example 13 Glucose Formation by Bacillus subtilis Alpha Amylases

In this example, experiments were conducted to determine the ability ofBacillus subtilis alpha amylases to convert maltose to glucose at pH 4.5and 5.6 using the glucose formation assay described in Example 2. Thereactions were analyzed after 2, 5 and 8 days.

As shown in FIG. 13, B. subtilis AmyE full length (SEQ ID NO: 1), AmyEtruncated (SEQ ID NO: 2), and variant α-amylase Amy 31A (SEQ ID NO: 3)effectively converted maltose to glucose, whereas the truncatedGeobacillus stearothermophilus α-amylase, AmyS (SEQ ID NO: 4), showedonly a minimal amount of glucose formation under these conditions.

Example 14 AmyE Action on Raw Starch

In this example, experiments were conducted to determine the ability offull-length AmyE (SEQ ID NO: 1), to hydrolyze insoluble granular(uncooked) starch. The HPLC method used for detection of saccharidesproduced from insoluble starch is as follows.

Purified Amy E (24.5 g/L) was diluted to a final concentration of 20.4ppm in malic acid buffer, pH 5.6. The protein was then added to a 5%corn flour solution in malic acid buffer, pH 5.6 to a finalconcentration of 1 ppm. The mixture was then incubated in a shaker at32° C. Samples were periodically removed and diluted into 50 mM NaOH toquench the reaction. 10 μL samples were then injected into an HPLCsystem (Agilent 1000) enabled with electrochemical detection. A PA1column was used with a gradient of NaOH and Na-acetate run at 25° C. Thedistribution was determined from previously run standards.

Results: Incubation of raw starch with 1 ppm of full-length AmyE enzymelead to a time-dependent release of numerous oligosaccharides (DP2, 3,4, 5, 6, 7) as well as glucose (DP1). The appearance of thesedegradation products was quantified by HPLC analysis of digestion timepoints. Data for 0, 30 and 90 minute samples is shown in FIG. 14.Comparable results were observed for the truncated AmyE enzyme (data notshown).

Example 15 Positional Libraries in Full Length AmyE

Positional libraries were generated at 295 sites in full-length AmyE(SEQ ID NO: 1) by Geneart (Geneart GmbH, Josef-Engert-strasse 11,D-93053 Regensburg, Germany). Table 3 lists each residue for which apositional library was made. Residues are numbered based on theirposition in SEQ ID NO: 1.

TABLE 3 Positional libraries generated in full-length AmyE VariantResidue number in Wild type No. full length AmyE residue 1 6 I 2 7 K 3 9G 4 10 T 5 11 I 6 12 L 7 13 H 8 14 A 9 15 W 10 16 N 11 17 W 12 19 F 1321 T 14 22 L 15 26 M 16 27 K 17 29 I 18 30 H 19 31 D 20 32 A 21 33 G 2234 Y 23 36 A 24 37 I 25 38 Q 26 39 T 27 40 S 28 41 P 29 42 I 30 43 N 3145 V 32 46 K 33 48 G 34 52 D 35 53 K 36 55 M 37 57 N 38 58 W 39 60 W 4061 L 41 62 Y 42 63 Q 43 64 P 44 65 T 45 66 S 46 67 Y 47 69 I 48 70 G 4971 N 50 72 R 51 74 L 52 75 G 53 77 E 54 79 E 55 80 F 56 81 K 57 82 E 5883 M 59 84 C 60 86 A 61 87 A 62 88 E 63 89 E 64 92 I 65 93 K 66 94 V 6795 I 68 96 V 69 97 D 70 98 A 71 99 V 72 100 I 73 101 N 74 102 H 75 103 T76 104 T 77 105 S 78 110 I 79 111 S 80 113 E 81 114 V 82 117 I 83 120 W84 121 T 85 122 H 86 126 Q 87 128 K 88 129 N 89 130 W 90 131 S 91 133 R92 135 D 93 136 V 94 137 T 95 138 Q 96 139 N 97 140 S 98 141 L 99 144 L100 145 Y 101 146 D 102 147 W 103 148 N 104 149 T 105 150 Q 106 151 N107 154 V 108 155 Q 109 157 Y 110 158 L 111 159 K 112 161 F 113 162 L114 164 R 115 165 A 116 167 N 117 168 D 118 169 G 119 170 A 120 171 D121 172 G 122 173 F 123 174 R 124 175 F 125 176 D 126 177 A 127 178 A128 179 K 129 180 H 130 181 I 131 182 E 132 183 L 133 184 P 134 186 D135 189 Y 136 191 S 137 193 F 138 194 W 139 196 N 140 197 I 141 198 T142 204 F 143 205 Q 144 206 Y 145 207 G 146 208 E 147 209 I 148 210 L149 211 Q 150 215 S 151 216 R 152 217 D 153 220 Y 154 223 Y 155 224 M156 225 D 157 226 V 158 227 T 159 228 A 160 229 S 161 230 N 162 231 Y163 232 G 164 235 I 165 236 R 166 237 S 167 238 A 168 239 L 169 241 N170 242 R 171 244 L 172 246 V 173 249 I 174 256 V 175 258 A 176 260 K177 261 L 178 262 V 179 263 T 180 264 W 181 265 V 182 266 E 183 267 S184 268 H 185 269 D 186 270 T 187 271 Y 188 272 A 189 273 N 190 278 S191 279 T 192 280 W 193 281 M 194 285 D 195 286 I 196 288 L 197 289 G198 290 W 199 291 A 200 292 V 201 293 I 202 294 A 203 295 S 204 296 R205 297 S 206 298 G 207 299 S 208 300 T 209 301 P 210 302 L 211 303 F212 304 F 213 305 S 214 306 R 215 307 P 216 312 N 217 315 R 218 316 F219 322 I 220 326 G 221 329 L 222 330 F 223 332 D 224 334 A 225 335 I226 336 T 227 337 A 228 338 V 229 339 N 230 340 R 231 341 F 232 342 H233 343 N 234 344 V 235 345 M 236 348 Q 237 350 E 238 351 E 239 352 L240 353 S 241 354 N 242 355 P 243 356 N 244 360 Q 245 361 I 246 362 F247 363 M 248 364 N 249 365 Q 250 366 R 251 370 G 252 371 V 253 372 V254 373 L 255 374 A 256 375 N 257 376 A 258 377 G 259 379 S 260 380 S261 381 V 262 383 I 263 384 N 264 387 T 265 389 L 266 391 D 267 392 G268 394 Y 269 396 N 270 397 K 271 398 A 272 399 G 273 402 S 274 403 F275 404 Q 276 405 V 277 407 D 278 408 G 279 409 K 280 410 L 281 411 T282 412 G 283 413 T 284 414 I 285 415 N 286 416 A 287 417 R 288 418 S289 419 V 290 420 A 291 421 V 292 422 L 293 423 Y 294 424 P 295 425 D

Twenty libraries at positions 27, 30, 45, 52, 75, 88, 89, 126, 131, 167,184, 223, 238, 241, 260, 307, 312, 344, 380 and 402 were generated inboth full-length AmyE and AmyE-tr. The B. subtilis transformantscontaining full-length AmyE substitution variants were cultured asdescribed in Example 8. Culture supernatants were used for assays.

Example 16 Performance of AmyE Variants

Using the procedures described in Examples 8 and 9, the relativeperformance or activity of full-length AmyE and truncated AmyE wascompared to the variants generated in full length AmyE and truncated AmyE. Table 4 summarizes the results of the site evaluation screens of AmyEfull length variants and Table 5 summarizes the results of the siteevaluation screens of AmyE truncated variants. Column 1 shows the aminoacid in the wildtype enzyme. Column 2 indicates the variant at theposition that was investigated in this study. The subsequent columnsshow the performance index values of the variants for the propertiestested. The properties tested were as follows: protein determination byBradford assay (expression), viscosity reduction rate (peak viscosity),reduction in post-liquefaction viscosity (final viscosity), degree ofpolymerization (iodine), reducing ends generated (reducing ends), totalglucose present (glucose), maltoheptahose hydrolysis at pH 5.8 (DP7 pH5.8), heat stability (30 min at 60° C.) using maltoheptahose hydrolysisat pH 5.8 (DP7 pH 5.8 heated), maltoheptaose hydrolysis at pH 4 (DP7 pH4), heat stability (30 min at 60° C.) using maltotriose hydrolysis at pH5.8 (DP3 HS), specific activity on corn flour substrate for 30 min (cornflour 30 min), rice starch stain microswatch assay at pH 8 (Cleaning pH8), and rice starch stain microswatch assay at pH 10 (cleaning, pH 10).Performance index (Pi) is defined as a ratio of performance of variantto parent protein

Lengthy table referenced here US08975056-20150310-T00001 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US08975056-20150310-T00002 Please refer tothe end of the specification for access instructions.

Based on the relative performance data of AmyE variants for theproperties described in Tables 2, 4 and 5, AmyE full-length andtruncated positions were classified as follows:

Fully restrictive positions: no neutral mutations for any propertytested

Non-fully restrictive positions: at least one neutral mutation for oneof the properties tested

Non-restrictive positions: ≧20% neutral mutations for at least oneproperty

Table 6 shows the non-fully restrictive positions and the identity ofthe wild-type amino acid residue at each position in the in thetruncated AmyE variants, along with the % neutral mutations for eachproperty. All of the positions listed may be mutated to alterperformance in the desired manner for any of the properties tested.Table 7 shows the non-fully restrictive positions and the wild-typeamino acid residues at each position in the full-length AmyE variants,with the % neutral mutations for each property. Again, all of thepositions listed may be mutated to alter performance in the desiredmanner for any of the properties tested.

TABLE 6 Non-fully restrictive positions in truncated AmyE Visc PAH DP7DP7 Clean Clean Peak BAH Unstressed pH 4 THER corn flour DP3 pH8 pH10Bradford PI PI Glucose PI PI PI Pi % HS Pi % Pi % Pi %PI >0.5% >0.5% >0.5% PI >0.5% >0.5% >0.5% >0.5% WT >0.5 >0.5 >0.5 >0.5ss2 ss2 ss2 ss2 ss2 ss2 ss2 POS AA ss1 ss1 ss1 ss1 TRUNC TRUNC TRUNCTRUNC TRUNC TRUNC TRUNC 1 L 100% 100% 100% 100% 100% 100% 100% 100% 100%100% 100% 2 T 100% 89% 100% 100% 84% 100% 100% 100% 100% 100% 100% 3 A100% 100% 89% 100% 79% 95% 95% 95% 95% 95% 95% 4 P 94% 88% 94% 100% 89%89% 89% 89% 89% 89% 89% 5 S 93% 93% 93% 86% 73% 100% 87% 87% 87% 87% 87%8 S 100% 100% 100% 100% 89% 100% 100% 100% 100% 100% 100% 18 S 100% 100%73% 82% 31% 56% 56% 56% 56% 50% 56% 20 N 100% 100% 100% 100% 89% 100%100% 100% 100% 100% 100% 23 K 100% 100% 100% 94% 83% 89% 89% 89% 89% 89%89% 24 H 100% 100% 100% 93% 85% 100% 100% 100% 100% 100% 100% 25 N 100%100% 100% 100% 75% 100% 100% 100% 100% 100% 100% 27 K 100% 100% 100% 94%94% 100% 100% 100% 100% 100% 100% 28 D 100% 100% 94% 100% 94% 100% 100%100% 100% 100% 100% 30 H 100% 89% 100% 100% 83% 94% 94% 94% 94% 94% 94%35 T 100% 100% 100% 100% 81% 100% 100% 100% 100% 100% 100% 44 Q 100%100% 100% 100% 68% 100% 100% 100% 100% 100% 100% 45 V 100% 69% 100% 46%22% 72% 72% 72% 72% 72% 61% 47 E 100% 100% 100% 100% 73% 100% 100% 100%100% 100% 100% 49 N 94% 100% 100% 82% 94% 100% 100% 100% 100% 100% 100%50 Q 100% 100% 94% 100% 94% 100% 100% 100% 100% 100% 100% 51 G 89% 94%100% 94% 78% 100% 100% 100% 100% 94% 89% 52 D 100% 79% 95% 95% 84% 100%100% 100% 100% 89% 100% 54 S 100% 94% 94% 94% 88% 100% 100% 100% 100%94% 100% 56 S 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 59Y 50% 81% 100% 19% 82% 94% 94% 65% 35% 41% 94% 68 Q 100% 100% 100% 94%83% 94% 94% 94% 89% 89% 94% 73 Y 57% 64% 93% 86% 11% 50% 44% 44% 44% 22%39% 76 T 100% 100% 86% 71% 79% 100% 100% 100% 100% 100% 100% 78 Q 100%100% 100% 100% 94% 100% 100% 94% 94% 94% 94% 85 A 100% 100% 100% 100%88% 88% 88% 88% 88% 88% 88% 88 E 100% 94% 94% 89% 61% 100% 100% 100%100% 100% 100% 89 E 100% 100% 100% 100% 95% 100% 100% 100% 100% 100%100% 90 Y 100% 100% 100% 100% 76% 88% 88% 88% 88% 88% 88% 91 G 76% 94%82% 82% 39% 78% 78% 78% 78% 67% 78% 106 S 59% 29% 100% 76% 5% 63% 63%63% 63% 53% 37% 107 Y 83% 67% 100% 89% 61% 100% 100% 100% 100% 100% 78%108 A 100% 100% 100% 100% 94% 94% 94% 94% 94% 94% 94% 109 A 100% 94%100% 83% 83% 100% 100% 100% 100% 100% 94% 112 N 100% 89% 100% 100% 95%100% 100% 100% 100% 100% 100% 115 K 82% 76% 88% 65% 32% 84% 84% 84% 84%84% 84% 116 S 100% 100% 100% 100% 94% 94% 94% 94% 94% 94% 94% 118 P 89%100% 100% 95% 83% 94% 94% 94% 94% 94% 94% 119 N 100% 80% 100% 93% 67%94% 83% 83% 83% 83% 83% 124 N 0% 0% 93% 64% 6% 47% 47% 47% 47% 24% 0%125 T 47% 35% 94% 88% 83% 100% 100% 100% 100% 100% 56% 126 Q 63% 81%100% 69% 100% 100% 100% 100% 94% 81% 75% 127 I 0% 0% 100% 0% 0% 21% 21%11% 16% 0% 0% 131 S 88% 88% 100% 94% 94% 88% 94% 94% 94% 94% 94% 132 D13% 7% 87% 33% 21% 53% 53% 53% 53% 42% 5% 134 W 0% 0% 92% 92% 38% 50%56% 56% 50% 50% 19% 142 L 43% 86% 57% 14% 76% 82% 82% 41% 12% 12% 71%143 G 0% 0% 8% 8% 7% 73% 73% 67% 67% 0% 0% 152 T 100% 95% 100% 100% 100%100% 100% 100% 100% 100% 100% 153 Q 95% 95% 95% 95% 95% 100% 100% 95%95% 95% 95% 156 S 100% 94% 100% 89% 94% 94% 94% 94% 94% 94% 94% 160 R100% 100% 94% 100% 89% 95% 95% 95% 95% 95% 95% 163 D 100% 100% 100% 100%94% 94% 94% 94% 94% 94% 94% 166 L 100% 100% 100% 94% 25% 94% 94% 94% 94%88% 94% 167 N 100% 100% 100% 100% 94% 94% 94% 94% 89% 89% 89% 184 P 6%0% 89% 33% 0% 37% 37% 37% 37% 26% 11% 185 D 20% 0% 100% 40% 0% 17% 17%17% 17% 6% 0% 187 G 5% 5% 95% 95% 63% 100% 100% 100% 100% 100% 5% 188 S50% 56% 100% 94% 94% 94% 94% 94% 94% 94% 53% 190 G 58% 8% 100% 75% 28%67% 67% 61% 61% 61% 11% 192 Q 0% 0% 0% 0% 86% 93% 93% 93% 93% 93% 93%195 P 47% 24% 88% 94% 50% 100% 100% 100% 100% 100% 78% 199 N 94% 100%81% 63% 63% 100% 100% 94% 94% 94% 100% 200 T 89% 68% 89% 84% 16% 84% 84%84% 84% 79% 84% 201 S 100% 100% 100% 100% 89% 100% 100% 100% 100% 100%100% 202 A 93% 93% 87% 87% 5% 42% 37% 37% 37% 37% 37% 203 E 100% 100%100% 100% 44% 94% 94% 94% 94% 94% 94% 212 D 0% 0% 45% 36% 17% 50% 50%50% 50% 6% 17% 213 S 82% 94% 82% 88% 100% 100% 100% 100% 100% 100% 100%214 A 44% 56% 100% 88% 81% 94% 94% 94% 94% 94% 50% 218 A 100% 89% 100%100% 100% 100% 100% 100% 100% 100% 100% 219 A 29% 29% 94% 35% 44% 89%89% 89% 89% 89% 44% 221 A 77% 54% 85% 77% 17% 67% 61% 56% 56% 56% 39%222 N 0% 0% 0% 0% 92% 100% 100% 100% 100% 100% 92% 223 Y 0% 0% 0% 0% 21%42% 42% 42% 42% 42% 42% 233 H 95% 95% 100% 42% 89% 95% 95% 95% 95% 95%95% 234 S 100% 94% 100% 100% 94% 94% 94% 94% 94% 94% 94% 238 A 100% 100%94% 100% 32% 95% 95% 95% 95% 89% 89% 240 K 100% 100% 100% 94% 89% 95%95% 89% 89% 89% 95% 241 N 100% 100% 100% 100% 94% 100% 100% 94% 94% 94%100% 243 N 100% 100% 100% 100% 95% 95% 95% 95% 95% 95% 95% 245 G 100%100% 93% 100% 81% 94% 94% 94% 94% 94% 94% 247 S 87% 87% 87% 87% 61% 72%67% 61% 61% 61% 67% 248 N 0% 0% 0% 0% 94% 94% 94% 94% 94% 94% 94% 250 S94% 94% 94% 100% 94% 94% 94% 94% 94% 94% 94% 251 H 100% 100% 100% 100%93% 100% 100% 100% 100% 100% 100% 252 Y 76% 47% 82% 94% 12% 47% 47% 47%47% 47% 24% 253 A 100% 88% 100% 94% 94% 100% 100% 100% 100% 94% 100% 254S 79% 57% 100% 79% 50% 93% 93% 86% 86% 64% 50% 255 D 0% 0% 0% 0% 78% 94%94% 94% 94% 94% 17% 257 S 0% 0% 0% 0% 76% 100% 100% 100% 100% 100% 100%259 D 93% 100% 93% 87% 53% 100% 100% 100% 100% 100% 100% 260 K 100% 100%83% 94% 37% 79% 79% 79% 79% 79% 79% 274 D 94% 100% 100% 100% 100% 100%100% 100% 100% 0% 100% 275 D 95% 100% 100% 89% 100% 100% 100% 74% 100%11% 100% 276 E 95% 89% 95% 79% 89% 95% 89% 84% 89% 37% 89% 277 E 95% 89%100% 79% 100% 100% 100% 84% 100% 32% 100% 282 S 100% 100% 100% 100% 84%100% 100% 100% 100% 84% 100% 283 D 88% 94% 88% 94% 38% 100% 100% 94% 94%38% 94% 284 D 95% 95% 95% 100% 95% 100% 100% 95% 95% 95% 100% 287 R 89%100% 95% 100% 59% 88% 88% 82% 82% 82% 88% 307 P 80% 100% 100% 100% 0%16% 16% 16% 16% 5% 16% 308 E 100% 100% 100% 100% 95% 100% 100% 100% 100%89% 100% 309 G 81% 94% 63% 100% 53% 76% 76% 76% 76% 53% 76% 310 G 31%94% 88% 81% 7% 67% 67% 67% 67% 0% 60% 311 G 95% 95% 84% 95% 11% 83% 83%83% 83% 0% 83% 312 N 95% 100% 100% 89% 94% 100% 94% 94% 94% 94% 94% 313G 84% 100% 100% 100% 72% 100% 100% 100% 100% 83% 100% 314 V 93% 93% 100%93% 93% 100% 100% 93% 93% 93% 87% 317 P 100% 100% 100% 89% 94% 100% 100%100% 100% 28% 100% 318 G 93% 93% 87% 93% 87% 93% 93% 93% 93% 93% 93% 319K 100% 100% 100% 100% 90% 100% 90% 90% 90% 90% 90% 320 S 100% 100% 100%100% 44% 94% 94% 94% 94% 44% 94% 321 Q 100% 106% 106% 106% 94% 100% 100%100% 100% 94% 100% 323 G 83% 67% 33% 100% 0% 28% 17% 17% 17% 6% 17% 324D 100% 100% 95% 100% 74% 100% 100% 100% 100% 68% 100% 325 R 88% 100% 88%100% 69% 94% 94% 94% 94% 81% 94% 327 S 92% 83% 58% 92% 43% 57% 57% 57%57% 21% 57% 328 A 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%331 E 100% 100% 100% 100% 94% 94% 94% 94% 94% 94% 94% 333 Q 100% 100%100% 100% 100% 100% 100% 100% 94% 100% 100% 344 V 100% 100% 100% 100%94% 94% 94% 94% 94% 94% 94% 346 A 107% 107% 107% 107% 94% 100% 100% 100%100% 100% 100% 347 G 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%100% 349 P 100% 100% 100% 100% 89% 94% 94% 94% 94% 94% 94% 357 G 100%100% 100% 100% 95% 100% 100% 100% 100% 100% 100% 358 N 100% 100% 100%100% 89% 95% 95% 95% 95% 95% 95% 359 N 100% 100% 100% 100% 95% 100% 100%100% 100% 100% 100% 367 G 100% 100% 100% 100% 72% 100% 100% 100% 100%100% 100% 368 S 100% 100% 100% 100% 87% 100% 100% 100% 100% 100% 100%369 H 100% 100% 100% 100% 95% 100% 100% 100% 100% 100% 100% 378 S 100%100% 100% 100% 89% 95% 95% 95% 95% 95% 95% 380 S 100% 100% 100% 100%100% 100% 100% 100% 100% 100% 100% 382 S 100% 100% 100% 100% 100% 100%100% 100% 100% 100% 100% 385 T 100% 100% 100% 100% 100% 100% 100% 100%100% 100% 95% 386 A 100% 100% 100% 93% 100% 100% 100% 100% 100% 100%100% 388 K 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 390 P100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 393 R 100% 100%100% 100% 100% 100% 100% 100% 100% 100% 100% 395 D 100% 95% 100% 100%94% 94% 94% 94% 94% 94% 94% 400 A 100% 100% 100% 100% 100% 100% 100%100% 100% 100% 100% 401 G 100% 100% 100% 100% 89% 100% 100% 100% 100%100% 74% 402 S 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%406 N 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

TABLE 7 Non-fully restrictive positions in full-length AmyE Visc PAH DP7Peak BAH DP7 pH 4 Cleaning 8 Cleaning THER Bradford PI PI GlucoseUnstressed PI PI 10 PI PI >0.5% >0.5% >0.5% PI >0.5%PI >0.5% >0.5% >0.5% PI >0.5% >0.5% WT ss2 ss2 ss2 ss2 ss2 ss2 ss2 ss2ss2 POS AA FULL FULL FULL FULL FULL FULL FULL FULL FULL 6 I 88% 100%100% 100% 100% 100% 100% 100% 100% 7 K 44% 100% 100% 100% 100% 100% 100%100% 100% 9 G 82% 100% 100% 100% 100% 100% 100% 100% 100% 10 T 47% 60%60% 60% 60% 60% 60% 60% 60% 11 I 25% 56% 56% 50% 56% 50% 56% 56% 56% 12L 12% 18% 18% 18% 18% 18% 18% 18% 18% 13 H 18% 82% 47% 6% 6% 6% 82% 59%76% 14 A 22% 44% 33% 28% 33% 28% 44% 44% 44% 15 W 6% 13% 13% 6% 6% 0%13% 13% 13% 16 N 11% 72% 72% 50% 67% 0% 72% 67% 72% 17 W 0% 35% 24% 6%18% 6% 35% 29% 35% 19 F 11% 50% 50% 44% 50% 44% 50% 50% 50% 21 T 47% 82%82% 82% 82% 76% 82% 82% 76% 22 L 25% 38% 38% 38% 38% 38% 38% 38% 38% 26M 13% 31% 31% 31% 31% 31% 31% 31% 31% 27 K 93% 100% 100% 100% 100% 100%100% 100% 100% 29 I 19% 44% 44% 38% 38% 19% 44% 44% 44% 30 H 88% 100%100% 94% 100% 94% 100% 100% 100% 31 D 83% 94% 94% 94% 94% 94% 94% 94%94% 32 A 12% 71% 71% 53% 71% 47% 71% 71% 71% 33 G 35% 94% 82% 35% 65%41% 94% 88% 94% 34 Y 11% 26% 16% 11% 16% 11% 26% 21% 26% 36 A 44% 83%83% 83% 83% 83% 83% 83% 83% 37 I 13% 25% 19% 19% 19% 19% 25% 25% 25% 38Q 0% 59% 35% 0% 29% 0% 59% 47% 59% 39 T 24% 53% 53% 41% 47% 24% 53% 47%47% 40 S 0% 53% 47% 18% 41% 6% 53% 53% 41% 41 P 0% 37% 32% 0% 26% 0% 37%37% 37% 42 I 6% 28% 28% 17% 28% 28% 28% 28% 28% 43 N 29% 53% 53% 53% 53%53% 53% 53% 53% 45 V 0% 73% 73% 67% 73% 60% 73% 73% 67% 46 K 44% 94% 94%94% 94% 94% 94% 94% 72% 48 G 44% 94% 94% 88% 88% 0% 94% 94% 56% 52 D 89%100% 100% 94% 100% 89% 100% 100% 100% 53 K 72% 94% 94% 94% 94% 94% 94%94% 94% 55 M 60% 93% 93% 87% 93% 47% 93% 93% 93% 57 N 47% 100% 100% 94%100% 41% 100% 100% 94% 58 W 6% 17% 17% 0% 0% 0% 17% 6% 17% 60 W 20% 93%93% 27% 7% 0% 93% 87% 20% 61 L 7% 57% 50% 36% 50% 21% 57% 57% 57% 62 Y6% 94% 6% 0% 0% 0% 89% 28% 78% 63 Q 17% 67% 50% 0% 0% 0% 67% 44% 56% 64P 11% 56% 44% 0% 17% 6% 56% 56% 50% 65 T 17% 61% 61% 61% 61% 61% 61% 61%61% 66 S 41% 94% 94% 94% 94% 94% 94% 94% 94% 67 Y 60% 100% 100% 87% 100%80% 100% 100% 87% 69 I 12% 35% 35% 24% 35% 24% 35% 35% 35% 70 G 0% 31%31% 13% 31% 6% 31% 31% 31% 71 N 0% 7% 7% 7% 7% 7% 7% 7% 7% 72 R 89% 100%100% 100% 100% 100% 100% 100% 79% 74 L 13% 44% 44% 31% 44% 25% 44% 44%44% 77 E 59% 88% 88% 82% 76% 82% 88% 88% 59% 79 E 33% 60% 60% 60% 60%53% 60% 60% 60% 80 F 6% 28% 22% 22% 22% 22% 28% 28% 28% 81 K 78% 94% 94%94% 94% 89% 94% 94% 94% 82 E 81% 94% 94% 94% 94% 94% 94% 94% 94% 83 M19% 38% 38% 38% 38% 38% 38% 38% 38% 84 C 21% 63% 63% 58% 63% 53% 63% 63%63% 86 A 88% 94% 94% 88% 94% 88% 94% 94% 94% 87 A 6% 24% 24% 12% 12% 12%24% 24% 18% 88 E 56% 89% 89% 83% 89% 83% 89% 89% 89% 89 E 94% 100% 100%100% 100% 94% 100% 100% 100% 92 I 17% 28% 28% 22% 28% 22% 28% 28% 22% 93K 44% 100% 100% 100% 100% 100% 100% 100% 100% 94 V 6% 29% 29% 24% 29%24% 29% 29% 29% 95 I 28% 33% 33% 33% 33% 33% 33% 33% 28% 96 V 6% 39% 39%39% 39% 39% 39% 39% 33% 98 A 6% 41% 41% 41% 41% 24% 41% 41% 35% 99 V 6%71% 18% 0% 6% 0% 71% 29% 71% 100 I 24% 53% 53% 47% 47% 41% 53% 53% 53%103 T 24% 41% 41% 41% 41% 29% 41% 41% 24% 104 T 11% 16% 16% 16% 16% 5%16% 16% 11% 105 S 50% 94% 94% 31% 56% 13% 94% 94% 94% 110 I 6% 17% 17%11% 11% 11% 17% 17% 6% 111 S 72% 100% 94% 94% 94% 89% 100% 94% 100% 113E 11% 89% 89% 83% 89% 72% 89% 89% 89% 114 V 13% 40% 33% 33% 7% 33% 33%33% 7% 117 I 5% 42% 42% 37% 42% 32% 42% 42% 16% 121 T 6% 22% 22% 17% 22%11% 22% 17% 6% 122 H 0% 6% 6% 6% 6% 0% 6% 6% 0% 126 Q 100% 100% 100%100% 94% 83% 100% 100% 67% 128 K 83% 94% 94% 94% 94% 94% 94% 94% 28% 129N 18% 88% 88% 88% 88% 88% 88% 88% 6% 130 W 31% 94% 94% 94% 81% 81% 94%94% 6% 131 S 94% 94% 94% 94% 94% 94% 94% 94% 83% 135 D 6% 41% 41% 12%41% 6% 41% 41% 18% 136 V 6% 38% 38% 19% 38% 13% 38% 38% 6% 138 Q 0% 56%31% 6% 19% 0% 56% 38% 6% 139 N 0% 41% 41% 35% 41% 35% 41% 41% 0% 140 S5% 26% 16% 11% 16% 5% 26% 16% 5% 141 L 58% 95% 21% 0% 0% 0% 79% 26% 21%144 L 78% 89% 6% 0% 0% 0% 72% 22% 6% 145 Y 6% 6% 6% 6% 6% 6% 6% 6% 6%146 D 0% 22% 6% 0% 0% 0% 22% 0% 17% 147 W 13% 50% 44% 31% 44% 25% 50%50% 25% 148 N 0% 50% 50% 50% 50% 50% 50% 50% 38% 149 T 0% 6% 6% 6% 6% 6%6% 6% 0% 150 Q 6% 25% 25% 25% 25% 25% 25% 25% 0% 151 N 63% 89% 89% 89%89% 84% 89% 89% 74% 154 V 6% 53% 35% 24% 35% 24% 53% 29% 47% 155 Q 6%75% 75% 75% 75% 75% 75% 75% 63% 157 L 11% 44% 44% 44% 39% 44% 44% 44%39% 158 Y 29% 53% 53% 53% 53% 53% 53% 53% 53% 159 K 28% 89% 89% 89% 89%89% 89% 89% 89% 161 F 6% 63% 63% 56% 44% 56% 63% 63% 25% 162 L 13% 44%44% 44% 44% 25% 44% 44% 44% 164 R 80% 93% 93% 93% 93% 93% 93% 93% 87%165 A 39% 61% 61% 56% 56% 61% 61% 56% 50% 167 N 100% 100% 100% 100% 100%100% 100% 100% 100% 168 D 88% 94% 88% 88% 88% 88% 88% 88% 88% 169 G 0%11% 6% 0% 0% 0% 0% 11% 0% 170 A 13% 20% 20% 20% 20% 20% 20% 20% 20% 171D 53% 87% 87% 87% 87% 87% 87% 87% 87% 172 G 6% 31% 6% 6% 6% 6% 31% 31%25% 173 F 0% 24% 24% 24% 24% 24% 24% 24% 24% 174 R 12% 76% 0% 0% 0% 0%65% 41% 41% 175 F 6% 24% 24% 24% 24% 24% 24% 24% 24% 176 D 26% 84% 0% 0%0% 0% 63% 21% 37% 177 A 39% 56% 6% 0% 0% 0% 44% 11% 11% 178 A 0% 28% 28%22% 28% 11% 28% 28% 28% 179 D 63% 100% 69% 6% 6% 0% 100% 31% 6% 180 H19% 44% 6% 0% 0% 0% 44% 19% 13% 181 I 6% 31% 31% 6% 31% 6% 31% 31% 6%183 L 0% 11% 11% 6% 11% 11% 11% 11% 0% 184 P 0% 79% 79% 16% 63% 5% 79%79% 68% 186 D 6% 6% 6% 6% 6% 6% 6% 6% 6% 189 Y 13% 13% 13% 13% 13% 13%13% 13% 13% 191 S 0% 24% 12% 12% 6% 12% 18% 24% 0% 193 F 0% 21% 21% 11%21% 16% 21% 16% 11% 194 W 0% 7% 7% 7% 7% 7% 7% 7% 0% 196 N 79% 95% 95%95% 95% 95% 95% 95% 95% 197 I 17% 44% 44% 44% 44% 44% 44% 44% 44% 198 T31% 69% 69% 69% 69% 69% 69% 69% 50% 204 F 24% 59% 59% 47% 59% 41% 59%59% 59% 205 Q 37% 95% 95% 95% 95% 95% 95% 95% 84% 206 Y 0% 6% 6% 0% 6%0% 6% 6% 6% 207 G 6% 19% 19% 19% 19% 0% 19% 19% 19% 208 E 73% 100% 0% 0%0% 0% 40% 13% 13% 209 I 11% 32% 32% 21% 21% 21% 32% 32% 11% 210 L 88%100% 18% 12% 6% 6% 88% 6% 35% 211 Q 18% 71% 71% 47% 65% 12% 71% 71% 6%215 S 0% 22% 22% 17% 22% 17% 22% 22% 0% 216 R 0% 11% 11% 11% 11% 11% 11%11% 0% 217 D 41% 94% 94% 82% 82% 82% 94% 88% 12% 220 Y 0% 6% 6% 6% 6% 6%6% 6% 0% 223 Y 22% 61% 61% 61% 61% 44% 61% 61% 50% 224 M 6% 24% 24% 24%24% 24% 24% 24% 24% 225 D 94% 100% 100% 100% 100% 100% 100% 100% 83% 226V 13% 44% 44% 38% 44% 19% 44% 44% 25% 227 T 0% 41% 12% 6% 6% 6% 41% 29%0% 228 A 0% 53% 53% 20% 47% 13% 53% 53% 13% 229 S 27% 87% 87% 7% 27% 7%87% 47% 33% 230 N 87% 100% 100% 93% 100% 87% 100% 93% 40% 231 Y 12% 41%41% 35% 41% 24% 41% 41% 12% 232 G 6% 39% 33% 0% 28% 0% 39% 39% 11% 235 I11% 33% 33% 28% 33% 22% 33% 33% 28% 236 R 41% 82% 82% 76% 82% 65% 82%82% 71% 237 S 89% 100% 100% 100% 100% 100% 100% 100% 94% 238 A 28% 94%94% 94% 94% 89% 94% 94% 94% 239 L 13% 38% 31% 25% 31% 19% 38% 38% 38%241 N 94% 100% 100% 94% 100% 94% 100% 100% 100% 242 R 94% 94% 94% 94%94% 94% 94% 94% 94% 244 L 22% 44% 44% 44% 44% 44% 44% 44% 44% 246 V 89%100% 100% 100% 100% 100% 100% 100% 100% 249 I 35% 71% 59% 53% 59% 53%71% 53% 47% 256 V 20% 40% 40% 40% 40% 40% 40% 40% 33% 258 A 47% 95% 95%95% 95% 95% 95% 89% 84% 260 K 56% 83% 83% 83% 83% 83% 83% 83% 83% 261 L6% 50% 50% 44% 50% 28% 50% 44% 33% 262 V 7% 36% 36% 21% 36% 14% 36% 36%36% 263 T 29% 47% 47% 41% 47% 41% 47% 47% 29% 264 W 88% 94% 56% 0% 6% 6%88% 50% 44% 265 V 0% 43% 43% 21% 36% 14% 43% 43% 36% 267 S 22% 0% 44% 6%28% 0% 67% 33% 39% 268 H 65% 94% 0% 0% 0% 6% 88% 12% 24% 269 D 94% 47%0% 12% 0% 0% 0% 12% 0% 270 T 81% 94% 81% 19% 63% 31% 94% 81% 94% 271 Y6% 6% 6% 6% 6% 6% 6% 6% 6% 272 A 25% 50% 50% 13% 44% 13% 50% 50% 38% 273N 83% 100% 72% 0% 28% 11% 100% 78% 89% 278 S 0% 17% 17% 6% 17% 0% 17%17% 17% 279 T 65% 100% 100% 29% 100% 12% 100% 100% 76% 280 W 13% 94% 94%94% 94% 0% 94% 94% 69% 281 M 13% 20% 20% 20% 20% 13% 20% 20% 20% 285 D11% 72% 72% 72% 72% 67% 72% 72% 67% 286 I 22% 44% 44% 44% 44% 28% 44%44% 44% 288 L 27% 40% 40% 40% 40% 33% 40% 40% 40% 289 G 6% 0% 11% 11%11% 0% 11% 11% 11% 290 W 6% 22% 22% 22% 22% 22% 22% 22% 22% 291 A 26%53% 53% 53% 53% 53% 53% 53% 53% 292 V 38% 50% 50% 50% 50% 50% 50% 50%50% 293 I 14% 64% 64% 43% 57% 43% 64% 64% 64% 294 A 21% 37% 37% 32% 37%32% 37% 37% 37% 295 S 12% 24% 24% 24% 24% 24% 24% 24% 24% 296 R 0% 41%41% 18% 41% 18% 41% 41% 24% 297 S 100% 100% 100% 100% 100% 100% 100%100% 100% 298 G 94% 94% 94% 94% 94% 94% 94% 94% 89% 299 S 41% 82% 82%82% 82% 71% 82% 82% 82% 300 T 22% 78% 78% 78% 78% 78% 78% 78% 61% 301 P47% 87% 87% 87% 80% 87% 87% 87% 67% 302 L 37% 63% 58% 21% 47% 26% 58%63% 32% 303 F 5% 21% 21% 21% 21% 21% 21% 21% 21% 304 F 20% 47% 27% 20%20% 7% 47% 40% 47% 305 S 19% 38% 38% 38% 38% 25% 38% 38% 31% 307 P 13%47% 27% 27% 27% 0% 47% 47% 47% 312 N 94% 94% 94% 94% 94% 88% 94% 94% 88%315 R 40% 93% 93% 53% 87% 7% 93% 93% 73% 316 R 12% 41% 47% 29% 47% 18%47% 47% 29% 322 I 17% 17% 17% 17% 17% 17% 17% 17% 11% 326 G 0% 11% 11%5% 5% 0% 11% 11% 11% 329 L 40% 80% 80% 80% 80% 73% 80% 80% 67% 330 F 11%42% 42% 42% 42% 42% 42% 42% 42% 332 D 41% 88% 88% 88% 76% 88% 88% 88%65% 334 A 100% 100% 100% 100% 100% 100% 100% 100% 100% 335 I 12% 35% 35%35% 29% 35% 35% 35% 35% 336 T 89% 94% 89% 89% 89% 89% 94% 89% 83% 337 A57% 100% 93% 86% 86% 86% 100% 100% 93% 338 V 44% 63% 63% 56% 56% 56% 63%63% 63% 339 N 6% 38% 38% 38% 38% 38% 38% 38% 38% 340 R 93% 93% 93% 93%93% 93% 93% 93% 93% 341 F 19% 44% 44% 44% 44% 44% 44% 44% 44% 342 H 31%81% 81% 81% 81% 81% 81% 81% 81% 343 N 89% 100% 100% 95% 95% 89% 100%100% 95% 344 V 89% 78% 94% 94% 94% 89% 89% 89% 89% 345 M 75% 94% 94% 94%94% 94% 94% 94% 94% 348 Q 88% 100% 100% 100% 100% 100% 100% 100% 94% 350E 6% 28% 28% 28% 28% 28% 28% 28% 22% 351 E 94% 100% 100% 94% 100% 94%100% 100% 100% 352 L 50% 100% 100% 94% 94% 94% 100% 100% 94% 353 S 94%94% 94% 94% 94% 94% 94% 94% 94% 354 N 0% 41% 41% 41% 41% 41% 41% 35% 41%355 P 44% 88% 88% 88% 88% 88% 88% 88% 88% 356 N 100% 100% 100% 100% 100%100% 100% 100% 100% 360 Q 100% 100% 100% 100% 100% 100% 100% 100% 100%361 I 50% 81% 81% 81% 81% 81% 81% 81% 81% 362 F 35% 71% 71% 71% 71% 71%71% 71% 71% 363 M 88% 94% 75% 69% 69% 81% 94% 88% 94% 364 N 56% 67% 67%67% 67% 67% 67% 67% 67% 365 Q 72% 94% 94% 89% 94% 89% 94% 89% 94% 366 R13% 75% 75% 63% 56% 63% 75% 75% 69% 370 G 0% 6% 6% 6% 6% 6% 6% 6% 6% 371V 53% 74% 74% 74% 74% 74% 74% 74% 68% 372 V 44% 63% 63% 63% 63% 63% 63%63% 56% 373 L 39% 67% 67% 67% 67% 67% 67% 67% 67% 374 A 41% 59% 59% 59%59% 59% 59% 59% 53% 375 N 65% 82% 82% 82% 82% 82% 82% 82% 82% 376 A 32%79% 79% 79% 79% 74% 79% 74% 74% 377 G 82% 94% 94% 88% 94% 88% 94% 88%94% 379 S 100% 88% 100% 100% 100% 100% 100% 100% 100% 380 S 94% 100%100% 100% 94% 94% 100% 100% 94% 381 V 94% 94% 94% 94% 94% 94% 94% 94%94% 383 I 47% 88% 88% 88% 88% 88% 88% 88% 88% 384 N 100% 100% 100% 100%100% 93% 100% 100% 100% 387 T 53% 73% 73% 73% 73% 67% 73% 73% 73% 389 L38% 88% 88% 81% 88% 81% 88% 88% 88% 391 D 100% 100% 100% 100% 100% 100%100% 100% 100% 392 G 94% 94% 94% 94% 94% 88% 94% 94% 88% 394 Y 58% 89%89% 89% 89% 89% 89% 89% 89% 396 N 19% 56% 56% 56% 56% 56% 56% 56% 56%397 K 100% 100% 100% 100% 100% 100% 100% 100% 94% 398 A 33% 94% 94% 94%94% 94% 94% 94% 94% 399 G 83% 94% 94% 94% 94% 89% 94% 94% 94% 402 S 94%94% 94% 94% 94% 94% 94% 94% 94% 403 F 44% 78% 78% 72% 72% 72% 78% 78%72% 404 Q 94% 100% 100% 100% 100% 100% 100% 100% 100% 405 V 50% 69% 75%75% 75% 75% 75% 75% 75% 407 D 89% 100% 100% 100% 94% 94% 100% 100% 100%408 G 89% 94% 100% 100% 94% 100% 100% 94% 100% 409 K 95% 95% 95% 95% 95%95% 89% 95% 89% 410 L 29% 65% 65% 53% 47% 53% 65% 65% 59% 411 T 95% 95%95% 95% 95% 95% 89% 95% 95% 412 G 35% 88% 88% 88% 88% 88% 88% 88% 82%413 T 100% 100% 100% 100% 100% 100% 100% 94% 94% 414 I 65% 82% 82% 82%82% 82% 82% 76% 82% 415 N 100% 100% 100% 100% 100% 100% 100% 100% 100%416 A 94% 100% 100% 100% 100% 100% 100% 100% 100% 417 R 73% 100% 100%100% 100% 100% 100% 100% 100% 418 S 87% 93% 93% 93% 93% 87% 93% 93% 93%419 V 72% 89% 83% 83% 83% 83% 83% 72% 83% 420 A 89% 94% 94% 94% 94% 94%94% 94% 89% 421 V 39% 44% 44% 44% 44% 44% 44% 44% 44% 422 L 53% 74% 74%74% 74% 74% 74% 74% 74% 423 Y 94% 94% 100% 100% 100% 100% 100% 94% 94%424 P 100% 94% 100% 100% 100% 100% 100% 100% 100% 425 D 100% 100% 100%100% 100% 100% 100% 100% 100%

As will be apparent from the foregoing description, certain positionscan be mutated in AmyE polypeptides to alter one or more propertieswithout substantially adversely affecting any one property. Thesemutations are collectively referred to as combinable mutations forsurface properties. Such positions are good candidates for making singlemutations, and combinations of mutations, since they are generallytolerant to manipulation. On the other hand, mutations at these positionimpart distinguishable properties on the resulting AmyE variants,indicating that they are important to enzyme structure and/or function.Corresponding positions in other parental amylases, including α-amylaseother than AmyE can be similarly mutated. In some cases, where thecorresponding position in another parental amylases includes a differentamino acid residue, it can be mutated to include an amino acid residuefound in a wild-type AmyE polypeptide. The positions are as follows:052D, 052E, 052I, 052K, 052L, 052N, 052Q, 052R, 052V, 056D, 056E, 056I,056K, 056L, 056N, 056Q, 056R, 056V, 089D, 089E, 089I, 089K, 089L, 089N,089Q, 089R, 089V, 152D, 152E, 152I, 152K, 152L, 152N, 152Q, 152R, 152V,153D, 153E, 153I, 153K, 153L, 153N, 153Q, 153R, 153V, 201D, 201E, 201I,201K, 201L, 201N, 201Q, 201R, 201V, 251D, 251E, 251I, 251K, 251L, 251N,251Q, 251R, 251V, 284D, 284E, 284I, 284K, 284L, 284N, 284Q, 284R, 284V,297D, 297E, 297I, 297K, 297L, 297N, 297Q, 297R, 297V, 308D, 308E, 308I,308K, 308L, 308N, 308Q, 308R, 308V, 321D, 321E, 321I, 321K, 321L, 321N,321Q, 321R, 321V, 328D, 328E, 328I, 328K, 328L, 328N, 328Q, 328R, 328V,347D, 347E, 347I, 347K, 347L, 347N, 347Q, 347R, 347V, 357D, 357E, 357I,357K, 357L, 357N, 357Q, 357R, 357V, 359D, 359E, 359I, 359K, 359L, 359N,359Q, 359R, 359V, 369D, 369E, 369I, 369K, 369L, 369N, 369Q, 369R, 369V,385D, 385E, 385I, 385K, 385L, 385N, 385Q, 385R, 385V, 388D, 388E, 388I,388K, 388L, 388N, 388Q, 388R, 388V, 391D, 391E, 391I, 391K, 391L, 391N,391Q, 391R, 391V, 400D, 400E, 400I, 400K, 400L, 400N, 400Q, 400R, 400V,416D, 416E, 416I, 416K, 416L, 416N, 416Q, 416R, and 416V. Mutations atall these mutations have PI values >0.5 for both protein and activity,demonstrating that the positions can be mutated without destroyingprotein expression or performance.

Example 17 Liquefaction in the Viscometer

Viscosity reduction of corn flour due to the action of the α-amylase wasmonitored using a HAAKE Viscotester 550 instrument. The substrate slurryis made up fresh daily in batch mode with 30% corn flour dry solids. ThepH was adjusted to 5.8 using sulfuric acid. 50 g of the slurry (15 g drysolids) is weighed out and pre-incubated, with stiffing, for 10 minutesto warm up to 70° C. Upon α amylase addition the temperature isimmediately ramped up from 70° C. to 85° C. with a rotation speed of 75rpm. Once the temperature of the slurry and enzyme mixture reaches 85°C., its temperature is held constant and viscosity is monitored for anadditional 30 minutes. The viscosity was measured throughout the run andis reported in μNm.

Wild-type AmyE (full-length or truncated) and several variants, thereof,were dosed at from about 0.25 to 1.5 mg/50 g of corn flour slurry andthe viscosity recorded.

A typical graph of the viscosity of the slurry over time is shown inFIG. 15, in which the wild-type full-length AmyE, and the AmyEfull-length variants I100F and W60M are compared. In other cases, onlythe peak and final viscosity of the slurry are tabulated. The resultsfor wild-type, full-length AmyE, and the variants L142F, L142G, L142Q,L142S, L142W, L142Y, A214I, A214V, S245Y, Q126F, Q126L, Q126P, Q126V,S131L, and S254I, made in the full-length AmyE background, are shown inTables 8 and 9. The results for wild-type, truncated AmyE, and thevariants W60L, W60M, W60N, I100F, I100M, S105M, S105W, G207A, T270A,T270E, T270L, T270N, T270V, and T279A, made in the truncated AmyEbackground, are shown in Table 10.

TABLE 8 Peak and final viscosity obtained using truncated wild-type andvariant AmyE (corn flour bag F) dose peak final (mg) viscosity viscosityWT 1.40 27300 1490 L142F 0.60 26100 550 L142G 0.30 29900 1925 L142Q 0.3031400 2800 L142S 0.25 30000 2495 L142W 1.40 33000 570 L142Y 1.20 278001940 A214I 1.40 24700 2330 A214V 1.40 21700 560 S245Y 1.40 25800 520Q126F 0.70 32300 2540 Q126L 1.40 30900 400 Q126P 1.40 25100 480 Q126V1.40 28300 520 S131L 1.40 26100 450

TABLE 9 Peak and final viscosity obtained using truncated wild-type andvariant AmyE (corn flour bag G) dose peak final (mg) viscosity viscosityWT 0.60 33600 890 L142F 0.45 28000 700 L142G 0.30 25300 2620 L142Q 0.3026300 4320 L142S 0.25 31200 11200 L142Y 0.60 28600 570 A214I 0.60 25200780 Q126F 0.50 32400 2020 S254I 0.60 32500 1320

TABLE 10 Peak and final viscosity obtained using truncated wild-type andvariant AmyE (corn flour bag H) dose peak final (mg) viscosity viscosityWT 1.40 29800 840 W60L 0.75 29200 1980 W60M 1.00 29100 2220 W60N 1.0030900 4250 I100F 0.75 29600 870 I100M 0.75 29200 840 S105M 0.75 303002170 S105W 0.75 30400 1960 G207A 0.75 29300 1920 T270A 0.75 31100 1540T270E 1.00 33200 1300 T270L 0.85 33000 1520 T270N 1.40 27700 560 T270V0.80 33900 2400 T279A 0.75 29400 1280

Improved performance in the viscometer assay can be identified using anumber of criteria, i.e., decreased peak viscosity, decreased finalviscosity, or a decreased enzyme dose required to produce similar peakor final viscosities relative to the dose required for a reference(control) enzyme. The bold highlighted text in Tables 8-10 indicate thecriteria in which each variant demonstrates improved performancecompared to the respective wild-type control.

Example 18 Thermostability of AmyE Full-Length, AmyE-Tr and AmyEVariants Using Differential Scanning Calorimetry

Excess heat capacity curves were measured using an ultrasensitivescanning high-throughput microcalorimeter, VP-Cap DSC (MicroCal, Inc.,Northampton, Mass., USA). The standard procedure for DSC measurementsand the theory of the technique is previously published (Freire, E.(1995) Differential Scanning calorimetry Methods. Mol. Biol.41:191-218). Approximately 500 μL of 0.5 mg/ml of AmyE-tr, or truncatedAmyE variants were scanned over 30-120° C. temperature range. The samesample was then re-scanned to check the reversibility of the process.The buffer used was 10 mM sodium acetate, pH 4.0 or pH 5.8. A 200° C./hrscan rate was used to minimize any artifacts that may result fromaggregation. The thermal midpoint (T_(m)) of the DSC curves was used asan indicator of the thermal stability. Table 11 shows the T_(m) valuesof wildtype truncated AmyE and truncated AmyE variants tested at pH 4.0and pH 5.8.

TABLE 11 T_(m) (in ° C.) of wild-type truncated AmyE and truncated AmyEvariants Variants T_(m) at pH 4.0 T_(m) at pH 5.8 wild type AmyE 69.974.9 truncated Q126L 73 78.1 Q126P 75 81 Q126V 70 75.6 L142F 74.2 79.1L142G 71 77.3 L142Q 70.1 75.7 L142W 74.1 79.6 L142Y 74.3 79.5 A214I 77.481.6 A214V 78 83 A214W 71 76.3

Example 19 Bread Staling

The following example relates to the use of an AmyE polypeptides toreduce bread staling.

A. Recipe for Baking Trials

Baking trials were carried out with a standard white bread sponge anddough recipe for US toast. The sponge dough is prepared from 1400 g offlour “Gold Medal” from General Mills, USA, 800 g of water, 40 g of rapeseed oil, 7.5 g GRINDSTED™ SSL P55 Veg, 10 g emulsifier DIMODAN™ PH200and 60 g of compressed yeast. The sponge is mixed for 1 min. at lowspeed and subsequently 3 min. at speed 2 on a Hobart spiral mixer. Thesponge is subsequently fermented for 3 hours at 25° C., 85% RH.

Thereafter, 600 g of “Gold Medal” flour, 18 g of compressed yeast, 5 gof calcium propionate, 160 g of sucrose, 5 g of calcium propionate, 432g of water and ascorbic acid (60 ppm final concentration) and ADA(azodicarbonamide; 40 ppm final concentration) are added to the sponge.The resulting dough is mixed for 1 min. at low speed and then 2 min. onhigh speed on a Diosna mixer. Then 30 g of salt is added to the dough.

The dough is rested for 5 min. at ambient temperature, and then 550 gdough pieces are scaled, moulded on Glimek sheeter with the settings1:4, 2:4, 3:15, 4:12 and width 8 on both sides and transferred to abaking form. After 65 min. proofing at 43° C. at 95% RH the doughs arebaked for 26 min. at 200° C. in an MIWE oven.

B. Protocol for Evaluation of Firmness, Resilience and Cohesiveness

Firmness, resilience and cohesiveness are determined by analysing breadslices by Texture Profile Analysis using a Texture Analyser From StableMicro Systems, UK. Calculation of firmness and resilience is doneaccording to preset standard supplied by Stable Micro System, UK. Theprobe used is aluminium 50 mm round.

Bread is sliced with the width of 12.5 mm. The slices are stamped out toa circular piece with a diameter of 45 mm and measured individually.

The following settings are used: Pre Test Speed: 2 mm/s, Test Speed: 2mm/s, Post Test Speed: 10 mm/s, Rupture Test Distance: 1%, Distance:40%, Force: 0.098 N, Time: 5.00 sec, Count: 5, Load Cell: 5 kg, TriggerType: Auto 0.01 N.

The mode of compression is a modification to the one used in Standardmethod AACC 74-09. The sample is compressed twice in the test.

C Protocol for Evaluation of Firmness

Firmness was determined at 40% compression during the first compression.The figure is the force needed to compress the slice to 40% of the totalthickness. The lower the value, the softer the bread. The firmness wasexpressed as a pressure, for example, in hPa.

D. Improved Handling Properties of Food Products Treated with an AmyEVariant Polypeptides

Bread was baked with 0.4 mg/kg of AmyE tr and the firmness of the breadis tested according to the protocol set out above at various times afterbaking. The firmness of the bread was tested. As a control, firmness ofbread baked without any enzyme is also measured.

FIG. 16 shows the results of a baking trial in which firmness of breadtreated with AmyE tr was compared to firmness of bread without enzyme. Areduced increase in firmness from day 1 to day 14 in the bread bakedwith AmyE tr indicated that the enzyme has antistaling effect and canimprove the fresh keeping of bread.

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US08975056B2). Anelectronic copy of the table will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. An isolated variant polypeptide havingalpha-amylase activity and at least one altered characteristic thatimproves enzyme performance, the variant polypeptide comprising: anamino acid sequence having at least 90% amino acid sequence identity toa parental alpha-amylase polypeptide selected from AmyE (SEQ ID NO: 1)or at least 92% amino acid sequence identity to a truncated variant ofAmyE (SEQ ID NO: 2), and a modification at one or more positionsselected from the group consisting of 35, 56, 65, 158, 189, 192, 298,320, 386 and 395, wherein the modification produces a variantpolypeptide having a performance index (PI) greater than 1.0 for atleast one characteristic that improves enzyme performance, and whereinthe modification at position 386 consists of the amino acid residuesubstitution A386R.
 2. The variant polypeptide of claim 1, comprising amodification at one or more positions selected from the group consistingof 35, 56, 65, 158, 189, 192, 298, 320, 386 and 395, wherein themodification produces a variant polypeptide having a performance index(PI) greater than 0.5 for protein expression, and a PI greater than 1.1for at least one characteristic that improves enzyme performance, andwherein the modification at position 386 consists of the amino acidresidue substitution A386R.
 3. The variant polypeptide of claim 1,wherein the one or more positions are selected from the group consistingof 35, 56, 65, 158, 189, 192, 298, 320, 386 and 395, which positions arenon-fully restrictive for performance in either the full-length ortruncated parental polypeptide, and wherein the modification at position386 consists of the amino acid residue substitution A386R.
 4. Thevariant polypeptide of claim 1, wherein the modification is asubstitution of one or more amino acid residues present in the parentalpolypeptide to different amino acid residues, at one or more positionsselected from the group consisting of T35V/I, S56W/R, T65I, L158V,Y189F, Q192N/E, G298L, S320T, and D395K.
 5. The variant polypeptide ofclaim 4, wherein the substitution changes the amino acid residue presentat position 35 to V or I, and the variant polypeptide exhibits increasedability to convert maltose and maltoheptaose substrates to glucosecompared to the parental polypeptide.
 6. The variant polypeptide ofclaim 4, wherein the substitution changes the amino acid residue presentat position 56 to W or R, and the variant polypeptide exhibits increasedability to convert maltose and maltoheptaose substrates to glucosecompared to the parental polypeptide.
 7. The variant polypeptide ofclaim 4, wherein the substitution changes the amino acid residue presentat position 65 to I, and the variant polypeptide exhibits increasedability to convert maltose and maltoheptaose substrates to glucosecompared to the parental polypeptide.
 8. The variant polypeptide ofclaim 4, wherein the substitution changes the amino acid residue presentat position 158 to V, and the variant polypeptide exhibits increasedability to convert maltose and maltoheptaose substrates to glucosecompared to the parental polypeptide.
 9. The variant polypeptide ofclaim 4, wherein the substitution changes the amino acid residue presentat position 189 to F, and the variant polypeptide exhibits increasedability to convert maltose and maltoheptaose substrates to glucosecompared to the parental polypeptide.
 10. The variant polypeptide ofclaim 4, wherein the substitution changes the amino acid residue presentat position 192 to N or E, and the variant polypeptide exhibitsincreased ability to convert maltose and maltoheptaose substrates toglucose compared to the parental polypeptide.
 11. The variantpolypeptide of claim 4, wherein the substitution changes the amino acidresidue present at position 298 to L, and the variant polypeptideexhibits increased ability to convert maltose and maltoheptaosesubstrates to glucose compared to the parental polypeptide.
 12. Thevariant polypeptide of claim 4, wherein the substitution changes theamino acid residue present at position 320 to T, and the variantpolypeptide exhibits increased ability to convert maltose andmaltoheptaose substrates to glucose compared to the parentalpolypeptide.
 13. The variant polypeptide of claim 1, wherein themodification is amino acid residue substitution A386R, and the variantpolypeptide exhibits increased ability to convert maltose andmaltoheptaose substrates to glucose compared to the parentalpolypeptide.
 14. The variant polypeptide of claim 4, wherein thesubstitution changes the amino acid residue present at position 395 toK, and the variant polypeptide exhibits increased ability to convertmaltose and maltoheptaose substrates to glucose compared to the parentalpolypeptide.
 15. The variant polypeptide of claim 1, wherein theparental polypeptide has at least 97% amino acid sequence identity tothe amino acid sequence of SEQ ID NO:
 3. 16. The variant polypeptide ofclaim 1, wherein the characteristic that improves enzyme performance isselected from the group consisting of increased thermal stability,increased specific activity, and increased protein expression.
 17. Adetergent composition comprising the variant polypeptide of claim
 1. 18.A starch conversion composition comprising the variant polypeptide ofclaim
 1. 19. The starch conversion composition of claim 18, furthercomprising an additional polypeptide having glucoamylase activity.